Novel Fab fragment libraries and methods for their use

ABSTRACT

The present invention provides Fab libraries and methods for using the Fab libraries to obtain antibodies against a target. The Fab library of the invention contains at least 10 9  different Fabs, and in some embodiments, at least 10 10  different Fabs. The Fab libraries of the invention are used to isolate polyclonal or monoclonal Fabs that bind with high specificity to targets.

[0001] This invention relates in general to phage display libraries ofhuman Fab fragments, and methods using the Fab fragment libraries toisolate high affinity antibodies. Especially, the invention relates topolynucleotides encoding a Fab library, Fab libraries, and methods fordesigning, constructing and selecting from Fab libraries.

[0002] Display on filamentous phage in combination with selection formsa powerful tool for the identification of peptide- or protein-baseddrugs (Winter et al., 1994; Clackson et al., 1994). Of these, antibodiesare especially of interest, due to their capacity to recognize a varietyof targets with high specificity and affinity. Particularly the use ofpartial or complete human antibodies, which elicit no or minimal immuneresponse when administered to patients, is yielding an increasing listof FDA-approved protein-based drugs (Holliger et al. 1998). Phagedisplay technology enables the generation of large repertoires of humanantibodies (Marks et al., 1991, Hoogenboom et al., 1992; Griffiths etal., 1993; Vaughan et al., 1996), and biopanning procedures permit theselection of individual antibodies with a desired specificity.

[0003] Key to the success of the technology were two criticalobservations: (i) the expression of functional antibody fragments bysecretion into the periplasm of E. coli (Better et al., 1988; Skerra etal., 1988), and, (ii) the rapid access to variable region gene pools bythe polymerase chain reaction (Larrick et al., 1989; Ward et al., 1989;Marks et al., 1991). For the construction of antibody libraries, V-genesare amplified from B-cell cDNA and heavy and light chain genes arerandomly combined and cloned to encode a combinatorial library ofsingle-chain Fv (scFv) or Fab antibody fragments (Marks et al., 1991;Clackson et al. 1991; Persson et al., 1991; Orum et al., 1993). Thenatural primary (unselected) antibody repertoire within B-cells containsa large array of antibodies that recognize a variety of antigens; thisarray can be cloned as a ‘naVve’ repertoire of rearranged genes, byharvesting the V-genes from the IgM mRNA of B-cells of unimmunized humandonors, isolated from peripheral blood lymphocytes (Marks et al., 1991),bone marrow or tonsils (Vaughan et al., 1996), or from similar animalsources (Gram et al., 1992). This procedure provides access toantibodies that have not yet encountered antigen, although the frequencyof those genuine ‘germline’ antibodies will depend heavily on the sourceof B-cells (Klein et al., 1997). A single ‘naïve’ library, ifsufficiently large and diverse, can indeed be used to generateantibodies to a large panel of antigens, including self, non-immunogenicand relatively toxic antigens (Griffiths et al., 1993; Marks et al.,1991). In a different approach, antibodies may be built artificially, byin vitro assembly of V-gene segments and D/J segments, yielding‘synthetic’ antibodies (Hoogenboom et al., 1992). A major drawback ofthese procedures is that from the initial ‘naVve’ and ‘synthetic’libraries, only moderate affinity antibodies were isolated (Marks etal., 1991; Nissim et al., 1994). Over the last few years more efficienttechniques have been developed to build larger libraries of antibodyfragments, using sophisticated in vivo recombination methods (Griffithset al., 1993) or brute force cloning procedures (Vaughan et al., 1996;Sheets et al., 1998). Such large libraries have yielded a greater numberof human antibodies per antigen tested, with an average much higheraffinity (up to sub-nanomolar). However, technical restrictions on thesize of libraries that may be obtained or handled in selection, the lossof library diversity upon library amplification, and the relatively longdown-stream analysis path of the selected antibodies, i.e., large scaleaffinity analysis, have limited the spread of these libraries as generictools in antibody generation.

[0004] Most large libraries made to date use the single chain format fordisplay on phage (Vaughan et al., 1996; Sheets et al. 1998). One reportdescribed the use of a human naive Fab library on phage (not permittingimmediate screening of selected soluble Fab fragments) (Griffiths etal., 1994). scFv's have the tendency to form dimers and higher ordermultimers in a clone-dependent and relatively unpredictable way(Weidner, et al. 1992; Holliger, et al. 1993; Marks et al., 1993). As aconsequence, the affinity assay used (such as BIAcore analysis) oftennecessitates purification of the selected antibody fragments. Forexample, ranking for off-rates using BIAcore is not easily possible withunpurified scFv fragments; the monomeric fraction of selected scFvclones first needs to be purified by affinity chromatography andgel-filtration (Sheets et al., 1998; Schier et al., 1996).

[0005] As was postulated and observed by Griffiths and colleagues(Griffiths et al., 1994), the size of the antibody library dictates theprobability of the selection of high affinity antibodies to the antigen.Comparison of the first naVve scFv repertoire containing 2.9×10⁷ clones(Marks et al., 1991), with a recently constructed scFv repertoire ofapproximately 1010 clones (Vaughan et al., 1996; Sheets et al. 1998),confirms this postulation: increasing the library size 500-fold resultedin approximately 100-fold higher affinities. This increase is caused bylowering the off-rates from 10⁻¹-10⁻² s⁻¹ for fragments selected fromthe smaller sized library to 10⁻³-10⁻⁴ s⁻¹ for those from the largerlibrary.

[0006] It is an object of the invention to create a Fab library that isa valuable source of antibodies for many different targets, and whichwill play a vital role in target discovery and validation in the area offunctional genomics.

[0007] The invention provides a plurality of polynucleotides encoding aFab library comprising a plurality of vector wherein the vectorcomprises:

[0008] a first and second cloning region, wherein

[0009] each cloning region comprises at least one, for the vectorunique, restriction enzyme cleavage site,

[0010] each cloning region being 5′ flanked by a ribosome binding siteand a signal sequence,

[0011] a polynucleotide encoding an anchor region, located 3′ of thesecond cloning region,

[0012] a first and a second plurality of variable polynucleotides,

[0013] each encoding a complete antibody variable region or part of anantibody variable region, possibly followed by a complete antibodyconstant region or part of an antibody constant region,

[0014] the first plurality of variable polynucleotides being cloned intothe vector at the restriction enzyme cleavage site(s) of the firstcloning region,

[0015] the second plurality of variable polynucleotides being clonedinto the vector at the restriction enzyme cleavage site(s) of the secondcloning region.

[0016] It is to be understood that the term “for the vector uniquerestriction enzyme cleavage site” refers to the presence of one of sucha restriction site in the vector sequence, without taking into accountthe possible presence of such a site on the above-mentioned first and/orsecond polynucleotides encoding a complete antibody variable region orpart of an antibody variable region, possibly followed by a completeantibody constant region or part of an antibody constant region. Thesaid first and second polynucleotides may comprise restriction sitesidentical to the “unique” site. This means that the said restrictionsite was “unique” before both first and second polynucleotide sequenceswere cloned into the vector.

[0017] The first and second variable polynucleotides are preferablycloned in the cloning region in a predetermined orientation. Therefore,in case the cloning region comprises a single unique restriction site,this site is preferably of such a type that non-identical restrictionends are generated, such as e.g., generated by the restriction enzymeSfiI. However, the cloning region may comprise two or more uniquerestriction sites, so that the variable polynucleotides can beconveniently cloned as a restriction fragment that has the correspondingends.

[0018] Preferably, in the vector according to the invention, the firstand second cloning regions, both ribosomal binding sites, signalsequences and the anchor sequence are part of a single polylinkersequence. Both cloning regions may therefore be part of a singlecassette, comprising the first cloning region, 5′ flanked by a ribosomalbinding site and a signal sequence, lying adjacent to the second cloningregion, also 5′ flanked by its corresponding ribosomal binding site anda signal sequence, and 3′ flanked by the anchor sequence.

[0019] Preferably, the first plurality of variable polynucleotides areV_(L) polynucleotides, and the second plurality of variablepolynuclotides are V_(H) polynucleotides. More preferably, the V_(L)polynucleotides are V₆ polynucleotides, V₆C₆ polynucleotides, V₈polynucleotides, V₈C₈ polynucleotides, a mixture of V₆ and V₈polynucleotides, or a mixture of V₆C₆ and V₈C₈ polynucleotides.

[0020] In another embodiment of the polynucleotides according to theinvention, the vector further comprises a tag for purification ordetection of an antibody, said tag for purification of the antibodypreferably comprising a poly-histidine tail; the tag for detection ofthe antibody is preferably a c-myc-derived tag.

[0021] In another embodiment of the polynucleotides according to theinvention, the vector further comprises an amber stop codon locatedbetween the second variable polynucleotide and the anchor protein.

[0022] In still another embodiment of the polynucleotides according tothe invention, the vector further comprises a C_(H1) domain locatedbetween the second variable polynucleotide and the anchor protein, theC_(H1) domain preferably being a human gamma-1 C_(H1) domain.

[0023] “Anchor protein” is defined as a protein or part thereof that canat least partially be accomodated in the outer coat of a particlegenerated by an organism expressing the library, such as a phage orvirus particle, or in the outer coat of an organism itself, in case theorganism itself expresses the library. The outer coat is herein definedas the structure of a cell, virus or phage particle defining the outersurface thereof. In case of a phage or phagemid expressing the library,the anchor protein may be a coat protein, such as the gene III product.However, other systems, known to the skilled person, may be used toobtain a library according to the present invention. Therefore, e.g.,transmembrane proteins, or the transmembrane domain thereof, may becontemplated to be used as anchor protein in eukaryotic expressionsystems. In the invention, the anchor protein may be fused to anantibody variable region or part thereof, resulting in the presentationof the said variable region to the outer environment of the organism,the region being anchored in its outer coat. In a preferred embodimentof the polynucleotides according to the invention, the anchor protein isa minor coat protein III of a filamentous phage f_(d).

[0024] In one embodiment of the invention, the polynucleotides accordingto the invention, and therefore the Fab library, encodes at least 10⁹different Fabs. In another embodiment of the invention, the Fab libraryof the invention encodes at least 10¹⁰ different Fabs. In still anotherembodiment of the invention, the Fab library encodes at least 3.7×10¹⁰different Fabs. In still another embodiment of the invention, the Fablibrary encodes 10⁹ to 3.7×10¹⁰ different Fabs.

[0025] Further, the invention provides a Fab library, comprising

[0026] a plurality of vectors as defined above,

[0027] the second cloning region in each vector forming a fusionpolynucleotide encoding a plurality of fusion proteins,

[0028] a plurality of capsid particles, wherein the plurality of vectorcontaining the first and second pluralities of variable polynucleotidesis packaged into the capsid particles, wherein

[0029] at least some of the capsid particles display the fusion proteinencoded by the vector packaged into the capsid on the surface of thecapsid.

[0030] Further the invention relates to a method of making a pluralityof polynucleotides encoding a Fab library, comprising the steps of:

[0031] amplifying a first plurality of variable polynucleotides with afirst set of primers,

[0032] amplifying a second plurality of variable polynucleotides with asecond set of primers,

[0033] wherein each set of primers comprises oligonucleotides designedto be homologous to the 5′ and 3′ end of variable polynucleotidesencoding antibody variable regions or parts thereof, such that they canbe used to amplify variable polynucleotide pools from natural orsynthetic sources of genes while retaining all or part of the antibody'santigen combining site;

[0034] cloning the first and second plurality of variablepolynucleotides into a plurality of vectors,

[0035] wherein the vector comprises:

[0036] a first and a second cloning region, wherein

[0037] each cloning region comprises at least one, for the vectorunique, restriction enzyme cleavage site,

[0038] each cloning region being 5′ flanked by a ribosome binding siteand a signal sequence,

[0039] a polynucleotide encoding an anchor region, located 3′ of thesecond cloning region,

[0040] wherein the first plurality of variable polynucleotides is clonedinto the restriction enzyme cleavage site(s) of the first cloning regionof the vector and the second plurality of variable polynucleotides intothe restriction enzyme cleavage site(s) of the second cloning region ofthe vector.

[0041] In one embodiment, the method of constructing the Fab librarycomprises the steps of: amplifying a plurality of variable gene poolswith a set of the primers, wherein the primers comprise oligonucleotidesdesigned to be homologous to the 5′ and 3′ end of variablepolynucleotides encoding antibody variable regions or parts thereof,such that they can be used to amplify variable polynucleotide pools fromnatural or synthetic sources of genes while retaining all or part of theantibody's antigen combining site; cloning the amplified variable genepools into a vector with a two-step procedure to obtain a Fab library;wherein the vector comprises a phage or phagemid vector which willaccommodate expression of the cloned antibody variable polynucleotidesas antibody Fab fragments, wherein one of the two antibody chains isfused to one of the phage coat proteins (e.g., geneIII product).

[0042] In one embodiment, the BACK primers were designed to have at themost three mutations in a total of twentyone to twentythree nucleotideswhen compared to the human germline gene segment region they would haveto bind to, but with at least 3 homologous residues towards the 3′ siteof the oligonucleotide. This set of olignucleotides will recogniseapproximately 90% of human germline gene segments and as such provideaccess to most of the present diversity of the B-cells in non-immunizedsources. In another embodiment, the heavy chain primers should end with‘GG’ to ensure stable binding at high annealing temperatures (at least55EC). Similarly the VkappaBACK primers and most of the VlambdaBACKprimers will be designed to preferentially end in ‘CC’. In an alternateembodiment, the primers consist of the sequences in FIG. 2.

[0043] The invention also provides methods for obtaining antibodiesspecific to an antigen from the Fab library. In certain embodiments, themethods of the invention allow a rapid initial screen of off-rates usingthe Fab libraries of the invention. In alternate embodiments, themethods of the patent are used to screen off-rates for a large series ofantigen specific Fabs using the Fab libraries of the invention.

[0044] The present invention also relates to isolated antibodiesspecific to an antigen of choice, and their corresponding nucleic acids,that are isolated from the Fab libraries of the invention. In analternative embodiment, these isolated antibodies are high affinityantibodies. The antibodies may be used as research reagents or astherapeutic products. The antibodies of the invention will be ideal forinvestigating the nature and localization of their targets, and theantibodies can be used to purify the target. Thus, the antibodies of theinvention will be important for target validation and target discoveryin the area of functional genomics.

[0045] The invention also relates to a vector as is defined above,comprising one of the first and one of the second plurality of thevariable polynucleotides cloned into the first and second cloning regionrespectively.

[0046] The present invention further relates to host cells containingthe Fab libraries of the invention or the polynucleotides that encodethe Fab libraries of the invention.

[0047] In one aspect the invention involves linking the desired specificbinding pair member, such as an antibody molecule, to a phage coatprotein. By then enriching for the specific binding pair member, such asby affinity techniques, for example, the DNA which encodes the specificbinding pair member is also enriched and may then be isolated. The DNAso obtained may then be cloned and expressed in other systems, yieldingpotentially large quantities of the desired specific binding pairmember, or may be subjected to sequencing and further cloning andgenetic manipulations prior to expression.

[0048] Typically the target for the specific binding pair member, e.g.,an antigen or hapten when the specific binding pair member is anantibody, is known, and the methods herein provide a means for creatingand/or identifying a specific binding pair member which specificallybinds the target of interest. Thus, when the protein is an antibody thepresent invention provides a novel means for producing antibodies,particularly monoclonal antibodies, with specificity for predeterminedtargets, thereby circumventing the laborious, time-consuming and oftenunpredictable process of conventional monoclonal antibody technology.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049]FIG. 1. Phagemid vector pCES1 for display of antibody Fabfragments. Schematic representation (A) and polylinker region (B) ofpCES1. The polylinker region comprises two signal sequences (‘S’; pelBand the geneIII leader sequence), the C6 domain, ribosome binding site(rbs), CH1 domain, hexa histidine tag (H6) and a c-myc derived sequence.Variable domain genes can be cloned as ApaLI-XhoI or ApaLI-Asc fragments(for VL or VLCL respectively) and SfiI/PstI-BstEII or SfiI-NotIfragments (for VH or VHCH1 respectively. The amber stop codon (*)between the antibody genes and bacteriophage gene III enables theproduction of soluble Fab fragments in a non-suppressor strain of E.coli. Expression of the bicistronic operon is under control of the LacZpromotor (pLacZ).

[0050]FIG. 2. This figure describes oligonucleotides used in oneembodiment for PCR amplification of human heavy and light chainV-regions.

[0051] The term “active” refers to those forms of the polypeptide whichretain the biologic and/or immunologic activities of any naturallyoccurring polypeptide.

[0052] The term “activated” cells as used in this application are thosewhich are engaged in extracellular or intracellular membranetrafficking, including the export of neurosecretory or enzymaticmolecules as part of a normal or disease process.

[0053] The term ‘antibody’ means an immunoglobulin whether natural orpartly or wholly synthetically produced. The term also covers anyprotein or polypeptide having a binding domain which is homologous to animmunoglobulin binding domain. These proteins can be derived fromnatural sources, or partly or wholly synthetically produced. Exampleantibodies are the immunoglobulin isotypes and the Fab, scFv, Fv, daab,VHH, Fd fragments.

[0054] The term ‘antibody polypeptide dimer’ means an association of twopolypeptide chain components of an antibody, capable of binding anantigen. Thus, it may be one arm of an antibody consisting of a heavychain and a light chain, it may be a Fab fragment consisting of V_(L),V_(H), C_(L) and C_(H1) antibody domains, or an Fv fragment consistingof a V_(L) domain and a V_(H) domain.

[0055] The term ‘capsid’ means a replicable genetic display package,with or without the genetic information. The capsids display a member ofa specific binding pair at its surface. The package may be a populationof bacteriophages which display an antigen binding domain, e.g., a Fab,at the surface of some or all of the capsids within the population. Thistype of package has been called a phage antibody (pAb).

[0056] The term ‘C_(H1) domain’ means the first constant region of theheavy chain of an antibody or part thereoff or extended with aminoacidsfrom the hinge regions as to allow pairing of the expressed (VH)CH1fragment with the antibody's light chain, and possible disulphide-bridgeformation. This may be the CH1 domain of a human antibody of isotypegamma-1.

[0057] A “component part of an antibody antigen-binding site” may be orcorrespond to a polypeptide chain component, e.g., a V_(H) or a V_(L)domain. However, it may be a CDR, or a V_(L) sequence plus CDR of aV_(H), a V_(H) sequence plus CDR of a V_(L), a V_(H) plus V_(L) sequencelacking only a CDR, and so on. The proviso is that the first and secondcomponent parts of an antigen-binding site of an antibody must incombination (together) form an antigen-binding site. Thus, if the secondcomponent part of an antigen-binding site of a non-human antibodyspecific for an antigen of interest is a CDR, then the first componentpart of an antigen-binding site of a human antibody will comprise theremainder of a V_(H) and V_(L) region required to form a antigen-bindingsite (with or without associated antibody constant domains (in a Fabformat), or with or without a linker peptide sequence (in a Fv format).The second component part of an antigen-binding site of a non-humanantibody may comprise a V_(L) domain plus part of a V_(H) domain, thatpart being one or more CDRs, for instance, perhaps CDR3. In such case,the first component part of an antigen-binding site of a human antibodywould comprise the remainder of a V_(H) sequence which in combinationwith the second component part forms an antigen-binding site. Of course,the converse situation holds and the person skilled in the art will beable to envisage other combinations of first and second component partswhich together form an antigen-binding site.

[0058] The term ‘conditionally defective’ means a gene which does notexpress a particular polypeptide under one set of conditions, butexpresses it under another set of conditions. An example, is a genecontaining an amber mutation expressed in non-suppressing or suppressinghosts respectively. Alternatively, a gene may express a protein orpolypeptide which is defective under one set of conditions, but notunder another set. An example is a gene with a temperature sensitivemutation.

[0059] The term “derivative” refers to polypeptides chemically modifiedby such techniques as ubiquitination, labeling (e.g., with radionuclidesor various enzymes), pegylation (derivatization with polyethyleneglycol) and insertion or substitution by chemical synthesis of aminoacids such as ornithine, which do not normally occur in human proteins.

[0060] The term ‘domain’ means a part of a protein or polypeptide thatis folded within itself and independently of other parts of the sameprotein or polypeptide and independently of a complementary bindingmember.

[0061] The term ‘eluant’ means a solution used to breakdown the linkagebetween two molecules. The linkage can be a non-covalent or covalentbond(s). The two molecules can be members of a sbp.

[0062] The term ‘expression modulating fragment,’ EMF, means a series ofnucleotides which modulates the expression of an operably linked ORF oranother EMF.

[0063] As used herein, a sequence is said to ‘modulate the expression ofan operably linked sequence’ when the expression of the sequence isaltered by the presence of the EMF. EMFs include, but are not limitedto, promoters, and promoter modulating sequences (inducible elements).One class of EMFs are fragments which induce the expression or anoperably linked ORF in response to a specific regulatory factor orphysiological event.

[0064] The term “Fab” refers to antibody fragments including fragmentswhich comprise two N-terminal portions of the heavy chain polypeptidejoined by at least one disulfide bridge in the hinge region and twocomplete light chain polypeptides, where each light chain is complexedwith one N-terminal portion of a heavy chain. Fab also includes Fabfragments which comprise all or a large portion of a light chainpolypeptide (e.g., V_(L)C_(L)) complexed with the N-terminal portion ofa heavy chain polypeptide (e.g., V_(H)C_(H1)).

[0065] The term “Fab library” refers to a collection of Fabpolynucleotide sequences within clones; or a genetically diversecollection of Fab polypeptides displayed on rgdps capable of selectionor screening to provide an individual Fab polypeptide or a mixedpopulation of Fab polypeptides.

[0066] The term ‘folded unit’ means a specific combination of analpha-helix and/or beta-strand and/or beta-turn structure. Domains andfolded units contain structures that bring together amino acids that arenot adjacent in the primary structure.

[0067] The term ‘genetically diverse population’ means antibodies orpolypeptide components thereof, this is referring not only to diversitythat can exist in the natural population of cells or organisms, but alsodiversity that can be created by artificial mutation in vitro or invivo. Mutation in vitro may for example, involve random mutagenesisusing oligonucleotides having random mutations of the sequence desiredto be varied. In vivo mutagenesis may for example, use mutator strainsof host microorganisms to harbour the DNA (see Example 38 of WO92/01047). The words “unique population” may be used to denote aplurality of e.g., polypeptide chains, which are not genetically diversei.e., they are all the same. A restricted population is one which isdiverse but less so than the full repertoire of an animal. The diversitymay have been reduced by prior selection, e.g., using antigen bindingspecificity.

[0068] The term ‘helper phage’ means a phage which is used to infectcells containing a defective phage genome and which functions tocomplement the defect. The defective phage genome can be a phagemid or aphage with some function encoding gene sequences removed. Examples ofhelper phages are M13KO7, M13K07 gene III no. 3; and phage displaying orencoding a binding molecule fused to a capsid protein.

[0069] The term ‘homologs’ means polypeptides having the same orconserved residues at a corresponding position in their primary,secondary or tertiary structure. The term also extends to two or morenucleotide sequences encoding the homologous polypeptides. Examplehomologous peptides are the immunoglobulin isotypes.

[0070] The term “host cell” refers to a prokaryotic or eukaryotic cellinto which the vectors of the invention may be introduced, expressedand/or propagated. Typical prokaryotic host cells include variousstrains of E. coli. Typical eukaryotic host cells are yeast orfilamentous fungi, or mammalian cells, such as Chinese hamster ovarycells, murine NIH 3t3 fibroblasts, or human embryonic kidney 193 cells.

[0071] The term ‘immunoglobulin superfamily’ means a family ofpolypeptides, the members of which have at least one domain with astructure related to that of the variable or constant domain ofimmunoglobulin molecules. The domain contains two B-sheets and usually aconserved disulphide bond (see A. F. Williams and A. N. Barclay 1988Ann. Rev Immunol. 6 381-405). Example members of an immunoglobulinsuperfamily are CD4, platelet derived growth factor receptor (PDGFR),intercellular adhesion molecule. (ICAM). Except where the contextotherwise dictates, reference to immunoglobulins and immunoglobulinhomologs in this application includes members of the immunoglobulinsuperfamily and homologs thereof.

[0072] The term “infection” refers to the introduction of nucleic acidsinto a suitable host cell by use of a virus or viral vector.

[0073] The term “intermediate fragment” means a nucleic acid between 5and 1000 bases in length, and preferably between 10 and 40 bp in length.

[0074] The term “isolated” as used herein refers to a nucleic acid orpolypeptide separated not only from other nucleic acids or polypeptidesthat are present in the natural source of the nucleic acid orpolypeptide, but also from polypeptides, and preferably refers to anucleic acid or polypeptide found in the presence of (if anything) onlya solvent, buffer, ion, or other component normally present in asolution of the same. The terms “isolated” and “purified” do notencompass nucleic acids or polypeptides present in their natural source.

[0075] The term ‘mutator strain’ means a host cell which has a geneticdefect which causes DNA replicated within it to be mutated with respectto its parent DNA. Example mutator strains are NR9046mutD5 and NR9046mut T1 (See Example 38 of WO 92/01047).

[0076] The term “naturally occurring polypeptide” refers to polypeptidesproduced by cells that have not been genetically engineered andspecifically contemplates various polypeptides arising frompost-translational modifications of the polypeptide including, but notlimited to, acetylation, carboxylation, glycosylation, phosphorylation,lipidation and acylation.

[0077] The term ‘nucleotide sequence’ refers to a heteropolymer ofnucleotides or the sequence of these nucleotides. The terms ‘nucleicacid’ and ‘polynucleotide’ are also used interchangeably herein to referto a heteropolymer of nucleotides. Generally, nucleic acid segmentsprovided by this invention may be assembled from fragments of the genomeand short oligonucleotide linkers, or from a series of oligonucleotides,or from individual nucleotides, to provide a synthetic nucleic acidwhich is capable of being expressed in a recombinant transcriptionalunit comprising regulatory elements derived from a microbial or viraloperon, or a eukaryotic gene.

[0078] The terms “oligonucleotide fragment” or a “polynucleotidefragment”, “portion,” or “segment” is a stretch of polypeptidenucleotide residues which is long enough to use in polymerase chainreaction (PCR) or various hybridization procedures to identify oramplify identical or related parts of mRNA or DNA molecules.

[0079] The terms “oligonucleotides” or “nucleic acid probes” areprepared based on the polynucleotide sequences provided in the presentinvention. Oligonucleotides comprise portions of such a polynucleotidesequence having at least about 15 nucleotides and usually at least about20 nucleotides. Nucleic acid probes comprise portions of such apolynucleotide sequence having fewer nucleotides than about 6 kb,usually fewer than about 1 kb. After appropriate testing to eliminatefalse positives, these probes may, for example, be used to determinewhether specific mRNA molecules are present in a cell or tissue or toisolate similar nucleic acid sequences from chromosomal DNA as describedby Walsh et al. (Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250).

[0080] The term “open reading frame,” ORF, means a series of nucleotidetriplets coding for amino acids without any termination codons and is asequence translatable into protein.

[0081] The term ‘phage vector’ means a vector derived by modification ofa phage genome, containing an origin of replication for a bacteriophage,but not one for a plasmid.

[0082] The term ‘phagemid vector’ means a vector derived by modificationof a plasmid genome, containing an origin of replication for abacteriophage as well as the plasmid origin of replication.

[0083] The term “phenotype” refers to a physical (e.g., pigment, or cellshape) and/or metabolic property of a cell which can be measured orexploited in some fashion and which is effected by the reporter gene.

[0084] The term ‘polylinker region’ means a polynucleotide that containsat least two restriction enzyme sites that are unique in the vector thatcontains the polylinker region, i.e., these restriction sites are easilyused cloning sites in the vector.

[0085] A polypeptide “fragment,” “portion,” or “segment” is a stretch ofamino acid residues of at least about 5 amino acids, often at leastabout 7 amino acids, typically at least about 9 to 13 amino acids, and,in various embodiments, at least about 17 or more amino acids. To beactive, any polypeptide must have sufficient length to display biologicand/or immunologic activity.

[0086] The term “probes” includes naturally occurring or recombinant orchemically synthesized single- or double-stranded nucleic acids. Theymay be labeled by nick translation, Klenow fill-in reaction, PCR orother methods well known in the art. Probes of the present invention,their preparation and/or labeling are elaborated in Sambrook, J. et al.,1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols inMolecular Biology, John Wiley & Sons, New York N.Y., both of which areincorporated herein by reference in their entirety.

[0087] The term “purified” as used herein denotes that the indicatednucleic acid or polypeptide is present in the substantial absence ofother biological macromolecules, e.g., polynucleotides, proteins, andthe like. In one embodiment, the polynucleotide or polypeptide ispurified such that it constitutes at least 95% by weight, morepreferably at least 99.8% by weight, of the indicated biologicalmacromolecules present (but water, buffers, and other small molecules,especially molecules having a molecular weight of less than 1000daltons, can be present).

[0088] The term “recombinant,” when used herein to refer to apolypeptide or protein, means that a polypeptide or protein is derivedfrom recombinant (e.g., microbial or mammalian) expression systems.‘Microbial’ refers to recombinant polypeptides or proteins made inbacterial or fungal (e.g., yeast) expression systems. As a product,‘recombinant microbial’ defines a polypeptide or protein essentiallyfree of native endogenous substances and unaccompanied by associatednative glycosylation. Polypeptides or proteins expressed in mostbacterial cultures, e.g., E. coli, will be free of glycosylationmodifications; polypeptides or proteins expressed in yeast will have aglycosylation pattern in general different from those expressed inmammalian cells.

[0089] The term ‘recombinant expression vehicle or vector’ refers to aplasmid or phage or virus or vector, for expressing a polypeptide from aDNA (RNA) sequence. An expression vehicle can comprise a transcriptionalunit comprising an assembly of (1) a genetic element or elements havinga regulatory role in gene expression, for example, promoters orenhancers, (2) a structural or coding sequence which is transcribed intomRNA and translated into protein, and (3) appropriate transcriptioninitiation and termination sequences. Structural units intended for usein yeast or eukaryotic expression systems preferably include a leadersequence enabling extracellular secretion of translated protein by ahost cell. Alternatively, where recombinant protein is expressed withouta leader or transport sequence, it may include an N-terminal methionineresidue. This residue may or may not be subsequently cleaved from theexpressed recombinant protein or polypeptide to provide a final product.

[0090] The term “recombinant expression system” means host cells whichhave stably integrated a recombinant transcriptional unit intochromosomal DNA or carry the recombinant transcriptional unitextrachromosomally. Recombinant expression systems as defined hereinwill express heterologous polypeptides or proteins upon induction of theregulatory elements linked to the DNA segment or synthetic gene to beexpressed. This term also means host cells which have stably integrateda recombinant genetic element or elements having a regulatory role ingene expression, for example, promoters or enhancers. Recombinantexpression systems as defined herein will express polypeptides orproteins endogenous to the cell upon induction of the regulatoryelements linked to the endogenous DNA segment or gene to be expressed.The cells can be prokaryotic or eukaryotic.

[0091] The term “recombinant variant” refers to any polypeptidediffering from naturally occurring polypeptides by amino acidinsertions, deletions, and substitutions, created using recombinant DNAtechniques. Guidance in determining which amino acid residues may bereplaced, added or deleted without abolishing activities of interest,such as cellular trafficking, may be found by comparing the sequence ofthe particular polypeptide with that of homologous peptides andminimizing the number of amino acid sequence changes made in regions ofhigh homology.

[0092] Preferably, amino acid “substitutions” are the result ofreplacing one amino acid with another amino acid having similarstructural and/or chemical properties, i.e., conservative amino acidreplacements. Amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

[0093] “Insertions” or “deletions” are typically in the range of about 1to 5 amino acids. The variation allowed may be experimentally determinedby systematically making insertions, deletions, or substitutions ofamino acids in a polypeptide molecule using recombinant DNA techniquesand assaying the resulting recombinant variants for activity.

[0094] Alternatively, where alteration of function is desired,insertions, deletions or non-conservative alterations can be engineeredto produce altered polypeptides. Such alterations can, for example,alter one or more of the biological functions or biochemicalcharacteristics of the polypeptides of the invention. For example, suchalterations may change polypeptide characteristics such asligand-binding affinities, interchain affinities, ordegradation/turnover rate. Further, such alterations can be selected soas to generate polypeptides that are better suited for expression, scaleup and the like in the host cells chosen for expression. For example,cysteine residues can be deleted or substituted with another amino acidresidue in order to eliminate disulfide bridges.

[0095] Alternatively, recombinant variants encoding these same orsimilar polypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsin the polynucleotide sequence may be reflected in the polypeptide ordomains of other peptides added to the polypeptide to modify theproperties of any part of the polypeptide, to change characteristicssuch as ligand-binding affinities, interchain affinities, ordegradation/turnover rate.

[0096] The term ‘repertoire of artificially rearranged immunoglobulingenes’ means a collection of nucleotide e.g., DNA, sequences derivedwholly or partly from a source other than the rearranged immunoglobulinsequences from an animal. This may include for example, DNA sequencesencoding VH domains by combining unrearranged V segments with D and Jsegments and DNA sequences encoding VL domains by combining V and Jsegments. Part or all of the DNA sequences may be derived byoligonucleotide synthesis.

[0097] The term ‘repertoire of rearranged immunoglobulin genes’ means acollection of naturally occurring nucleotides e.g., DNA sequences whichencoded expressed immunoglobulin genes in an animal. The sequences aregenerated by the in vivo rearrangement of e.g., V, D and J segments forH chains and e.g., the V and J segments for L chains. Alternatively thesequences may be generated from a cell line immunised in vitro and inwhich the rearrangement in response to immunisation occursintracellularly. The word “repertoire” is used to indicate geneticdiversity.

[0098] The term ‘replicable genetic display package’ (Rgdp) means abiological particle which has genetic information providing the particlewith the ability to replicate. The particle can display on its surfaceat least part of a polypeptide. The polypeptide can be encoded bygenetic information native to the particle and/or artificially placedinto the particle or an ancestor of it. The displayed polypeptide may beany member of a specific binding pair e.g., heavy or light chain domainsbased on an immunoglobulin molecule, an enzyme or a receptor etc. Theparticle may be a virus e.g., a bacteriophage such as fd or M13.

[0099] The term “reporter gene” refers to a nucleic acid which encodes aprotein or polypeptide that produces a phenotypic change in the hostcell that may be measured and/or used to separate host cells. Forexample, the reporter gene may encode a protein or polypeptide that hasflourescent properties, e.g., β-galactosidase, auto-fluorescent proteinGFP, etc.; or the reporter gene may encode a selectable marker, e.g.,antibiotic resistance; or an epitope that is expressed on the surface ofthe host cell.

[0100] The term ‘ribosome binding site’ means a polyribonucleotide thatallows a ribosome to select the proper initiation codon during theinitiation of translation. In some prokaryotes, this polyribonucleotideis called the Shine-Dalgarno sequence, and the Shine-Delgarno sequencebase pairs with the 16S RNA of the ribosome.

[0101] The term “secreted” protein or polypeptide refers to a protein orpolypeptide that is transported across or through a membrane, includingtransport as a result of signal sequences in its amino acid sequencewhen it is expressed in a suitable host cell. “Secreted” proteins orpolypeptides include without limitation proteins or polypeptidessecreted wholly (e.g., soluble proteins) or partially (e.g., receptors)from the cell in which they are expressed. “Secreted” proteins orpolypeptides also include without limitation proteins or polypeptideswhich are transported across the membrane of the endoplasmic reticulum.

[0102] The term ‘signal sequence’ means an amino acid sequence that isfound at the amino terminus of a polypeptide and directs transportationof the polypeptide across or through a membrane. Signal sequencesinclude amino terminal polypeptides that are 13-36 residues long, andhave a 7 to 13 residue hydrophobic core flanked by several hydrophilicresidues that usually include one or more basic residues near theN-terminus.

[0103] The term “stringent” is used to refer to conditions that arecommonly understood in the art as stringent. An exemplary set ofconditions include a temperature of 60-70° C., (preferably about 65° C.)and a salt concentration of 0.70 M to 0.80 M (preferably about 0.75M).Further exemplary conditions include, hybridizing conditions that (1)employ low ionic strength and high temperature for washing, for example,0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.; (2) employduring hybridization a denaturing agent such as formamide, for example,50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2× SSC and 0.1% SDS.

[0104] In instances wherein hybridization of deoxyoligonucleotides isconcerned, additional exemplary stringent hybridization conditionsinclude washing in 6× SSC/0.05% sodium pyrophosphate at 37EC (for14-base oligos), 4BEC (for 17-base oligos), 55EC (for 20-base oligos),and 60EC (for 23-base oligos).

[0105] As used herein, “substantially equivalent” can refer both tonucleotide and amino acid sequences, for example a mutant sequence, thatvaries from a reference sequence by one or more substitutions,deletions, or additions, the net effect of which does not result in anadverse functional dissimilarity between the reference and subjectsequences. Typically, such a substantially equivalent sequence variesfrom one of those listed herein by no more than about 20% (i.e., thenumber of individual residue substitutions, additions, and/or deletionsin a substantially equivalent sequence, as compared to the correspondingreference sequence, divided by the total number of residues in thesubstantially equivalent sequence is about 0.2 or less). Such a sequenceis said to have 80% sequence identity to the listed sequence. In oneembodiment, a substantially equivalent, e.g., mutant, sequence of theinvention varies from a listed sequence by no more than 10% (90%sequence identity); in a variation of this embodiment, by no more than5% (95% sequence identity); and in a further variation of thisembodiment, by no more than 2% (98% sequence identity). Substantiallyequivalent, e.g., mutant, amino acid sequences according to theinvention generally have at least 95% sequence identity with a listedamino acid sequence, whereas substantially equivalent nucleotidesequence of the invention can have lower percent sequence identities,taking into account, for example, the redundancy or degeneracy of thegenetic code. For the purposes of the present invention, sequenceshaving substantially equivalent biological activity and substantiallyequivalent expression characteristics are considered substantiallyequivalent. For the purposes of determining equivalence, truncation ofthe mature sequence (e.g., via a mutation which creates a spurious stopcodon) should be disregarded.

[0106] Nucleic acid sequences encoding such substantially equivalentsequences, e.g., sequences of the recited percent identities, canroutinely be isolated and identified via standard hybridizationprocedures well known to those of skill in the art.

[0107] The term ‘suppressible translational stop codon’ means a codonwhich allows the translation of nucleotide sequences downstream of thecodon under one set of conditions, but under another set of conditionstranslation ends at the codon. Example of suppressible translationalstop codons are the amber, ochre and opal codons.

[0108] The term ‘tag’ means an extension of the antibody Fab fragment,for example expressed at the carboxyterminus of the heavy chain, thatcomprises at least one amino acids but more typically five to fifteenamino acids, and that can be specifically recognised by an antibody orother binding ligand or binding matrix for the sequence. Tags may becombined in the same Fab. Examples are a stretch of five histidineresidues that can be recognised by specific antibodies and by definedimmobilised metal ions, and a stretch of the following 12 amino acids(EQKLISEEDLN)that are recognised by the 9E10 antibody (Marks et al.,1991).

[0109] The term ‘target’ means any molecule that is antigenic, e.g., canbe recognized with reasonably specificity by an antibody from the Fablibrary.

[0110] The term ‘target element’ refers to a nucleic acid sequence thatalters the expression of the target gene. Target elements include, butare not limited to, promoters, and promoter modulating sequences(inducible elements). One class of target elements are fragments whichinduce the expression in response to a specific regulatory factor orphysiological event.

[0111] The term “transfection” refers to the taking up of an expressionvector by a suitable host cell, whether or not any coding sequences arein fact expressed.

[0112] The term “transformation” means introducing DNA into a suitablehost cell so that the DNA is replicable, either as an extrachromosomalelement, or by chromosomal integration.

[0113] The term “universal set” refers to a set of nucleic acids, mostpreferably a set of oligonucleotides, which represent all possiblecombinations of sequence for a given length of nucleotides, e.g., all4096 insert oligonucleotides six nucleotides in length. In a preferredembodiment, the term universal set refers to the set of all possibleoligonucleotides of a given length, wherein one or more positions in theoligonucleotides are held constant (i.e., the same nucleotide is presentat this position in all members of the set).

[0114] As used herein, an ‘uptake modulating fragment,’ UMF, means aseries of nucleotides which mediate the uptake of a linked DNA fragmentinto a cell. UMFs can be readily identified using known UMFs as a targetsequence or target motif with the computer-based systems describedbelow.

[0115] The presence and activity of a UMF can be confirmed by attachingthe suspected UMF to a marker sequence. The resulting nucleic acidmolecule is then incubated with an appropriate host under appropriateconditions and the uptake of the marker sequence is determined. Asdescribed above, a UMF will increase the frequency of uptake of a linkedmarker sequence.

[0116] The term ‘vector’ refers to a plasmid or phage or virus orvector, for expressing a polypeptide from a DNA (RNA) sequence. Thevector can comprise a transcriptional unit comprising an assembly of (1)a genetic element or elements having a regulatory role in geneexpression, for example, promoters or enhancers, (2) a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and (3) appropriate translation initiation and terminationsequences. Structural units intended for use in yeast or eukaryoticexpression systems may include a leader sequence enabling extracellularsecretion of translated protein by a host cell.

[0117] The term ‘V_(L) polynucleotides’ means polynucleotides encodingthe CDR containing domains of some or all of the light chain genes fromthe V₆- and/or V₈- families.

[0118] The term ‘VH polynucleotides’ means polynucleotides encoding theCDR containing domains of some or all of the heavy chain genes from theheavy chain gene family.

[0119] Each of the above terms is meant to encompasses all that isdescribed for each, unless the context dictates otherwise.

[0120] The recombinant constructs of the present invention comprise avector, such as a plasmid or viral vector, into which a nucleic acid(s)of interest may be inserted. The vector may further comprise regulatorysequences, including for example, a promoter, operably linked to thenucleic acid(s) of interest. Large numbers of suitable vectors andpromoters are known to those of skill in the art and are commerciallyavailable for generating the recombinant constructs of the presentinvention. The following vectors are provided by way of example.Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a,pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG(Stratagene) pSVK3, PBPV, pMSG, pSVL (Pharmacia).

[0121] Methods which are well known to those skilled in the art can beused to construct vectors containing a polynucleotide of the inventionand appropriate transcriptional/translational control signals. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination. See, forexample, the techniques described in Maniatis et al., Molecular CloningA Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989) andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley Interscience, N.Y. (1989).

[0122] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P, and trc.Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.

[0123] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heatshock proteins, among others. The polynucleotide of the invention isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the polynucleotide of the inventioncan encode a fusion protein including an N-terminal identificationpeptide imparting desired characteristics, e.g., stabilization orsimplified purification of expressed recombinant product.

[0124] Useful expression vectors for bacteria are constructed byinserting a polynucleotide of the invention together with suitabletranslation initiation and termination signals, optionally in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0125] As a representative but nonlimiting example, useful expressionvectors for bacteria can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec,Madison, Wis., USA). These pBR322 ‘backbone’ sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0126] The present invention further provides host cells containing thevectors of the present invention, wherein the nucleic acid has beenintroduced into the host cell using known transformation, transfectionor infection methods. The host cell can be a higher eukaryotic hostcell, such as a mammalian cell, a lower eukaryotic host cell, such as ayeast cell, or the host cell can be a prokaryotic cell, such as abacterial cell. Introduction of the recombinant construct into the hostcell can be effected, for example, by calcium phosphate transfection,DEAE, dextran mediated transfection, or electroporation (Davis, L. etal., Basic Methods in Molecular Biology (1986)).

[0127] Any host/vector system can be used to identify one or more of thetarget elements of the present invention. These include, but are notlimited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells,and Sf9 cells, as well as prokaryotic host such as E. coli and B.subtilis. The most preferred cells are those which do not normallyexpress the particular reporter polypeptide or protein or whichexpresses the reporter polypeptide or protein at low natural level.

[0128] The host of the present invention may also be a yeast or otherfungi. In yeast, a number of vectors containing constitutive orinducible promoters may be used. For a review see, Current Protocols inMolecular Biology, Vol. 2, Ed. Ausubel et al., Greene Publish. Assoc. &Wiley Interscience, Ch. 13 (1988); Grant et al., Expression andSecretion Vectors for Yeast, in Methods in Enzymology, Ed. Wu &Grossman, Acad. Press, N.Y. 153:516-544 (1987); Glover, DNA Cloning,Vol. II, IRL Press, Wash., D.C., Ch. 3 (1986); Bitter, Heterologous GeneExpression in Yeast, in Methods in Enzymology, Eds. Berger & Kimmel,Acad. Press, N.Y. 152:673-684 (1987); and The Molecular Biology of theYeast Saccharomyces, Eds. Strathern et al., Cold Spring Harbor Press,Vols. I and II (1982).

[0129] The host of the invention may also be a prokaryotic cell such asE. coli, other enterobacteriaceae such as Serratia marescans, bacilli,various pseudomonads, or other prokaryotes which can be transformed,transfected, infected, etc. (i.e., a method exists for introducingnucleic acids to the host cell).

[0130] The present invention further provides host cells geneticallyengineered to contain the polynucleotides of the invention. For example,such host cells may contain nucleic acids of the invention introducedinto the host cell using known transformation, transfection or infectionmethods. The present invention still further provides host cellsgenetically engineered to express the polynucleotides of the invention,wherein such polynucleotides are in operative association with aregulatory sequence heterologous to the host cell which drivesexpression of the polynucleotides in the cell.

[0131] The host cell can be a higher eukaryotic host cell, such as amammalian cell, a lower eukaryotic host cell, such as a yeast cell, orthe host cell can be a prokaryotic cell, such as a bacterial cell.Introduction of the recombinant construct into the host cell can beeffected by calcium phosphate transfection, DEAE, dextran mediatedtransfection, or electroporation (Davis, L. et al., Basic Methods inMolecular Biology (1986)). The host cells containing one ofpolynucleotides of the invention, can be used in conventional manners toproduce the gene product encoded by the isolated fragment (in the caseof an ORF) or can be used to produce a heterologous protein under thecontrol of the EMF.

[0132] Any host/vector system can be used to express one or more of theORFs of the present invention. These include, but are not limited to,eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9cells, as well as prokaryotic host such as E. coli and B. subtilis. Themost preferred cells are those which do not normally express theparticular polypeptide or protein or which expresses the polypeptide orprotein at low natural level. Mature proteins can be expressed inmammalian cells, yeast, bacteria, or other cells under the control ofappropriate promoters. Cell-free translation systems can also beemployed to produce such proteins using RNAs derived from the DNAconstructs of the present invention. Appropriate cloning and expressionvectors for use with prokaryotic and eukaryotic hosts are described bySambrook, et al., in Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor, New York (1989), the disclosure of which ishereby incorporated by reference.

[0133] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celltines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements. Recombinant polypeptides and proteins produced inbacterial culture are usually isolated by initial extraction from cellpellets, followed by one or more salting-out, aqueous ion exchange orsize exclusion chromatography steps. Protein refolding steps can beused, as necessary, in completing configuration of the mature protein.Finally, high performance liquid chromatography (HPLC) can be employedfor final purification steps. Microbial cells employed in expression ofproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents.

[0134] A number of types of cells may act as suitable host cells forexpression of the protein. Mammalian host cells include, for example,monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1cells, other transformed primate cell lines, normal diploid cells, cellstrains derived from in vitro culture of primary tissue, primaryexplants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkatcells.

[0135] Alternatively, it may be possible to produce the protein in lowereukaryotes such as yeast or in prokaryotes such as bacteria. Potentiallysuitable yeast strains include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing heterologous proteins. Potentially suitablebacterial strains include Escherichia coli, Bacillus subtilis,Salmonella typhimurium, or any bacterial strain capable of expressingheterologous proteins. If the protein is made in yeast or bacteria, itmay be necessary to modify the protein produced therein, for example byphosphorylation or glycosylation of the appropriate sites, in order toobtain the functional protein. Such covalent attachments may beaccomplished using known chemical or enzymatic methods.

[0136] In another embodiment of the present invention, cells and tissuesmay be engineered to express an endogenous gene comprising thepolynucleotides of the invention under the control of inducibleregulatory elements, in which case the regulatory sequences of theendogenous gene may be replaced by homologous recombination. Asdescribed herein, gene targeting can be used to replace a gene'sexisting regulatory region with a regulatory sequence isolated from adifferent gene or a novel regulatory sequence synthesized by geneticengineering methods. Such regulatory sequences may be comprised ofpromoters, enhancers, scaffold-attachment regions, negative regulatoryelements, transcriptional initiation sites, regulatory protein bindingsites or combinations of said sequences. Alternatively, sequences whichaffect the structure or stability of the RNA or protein produced may bereplaced, removed, added, or otherwise modified by targeting, includingpolyadenylation signals. mRNA stability elements, splice sites, leadersequences for enhancing or modifying transport or secretion propertiesof the protein, or other sequences which alter or improve the functionor stability of protein or RNA molecules.

[0137] The targeting event may be a simple insertion of the regulatorysequence, placing the gene under the control of the new regulatorysequence, e.g., inserting a new promoter or enhancer or both upstream ofa gene. Alternatively, the targeting event may be a simple deletion of aregulatory element, such as the deletion of a tissue-specific negativeregulatory element. Alternatively, the targeting event may replace anexisting element; for example, a tissue-specific enhancer can bereplaced by an enhancer that has broader or different cell-typespecificity than the naturally occurring elements. Here, the naturallyoccurring sequences are deleted and new sequences are added. In allcases, the identification of the targeting event may be facilitated bythe use of one or more selectable marker genes that are contiguous withthe targeting DNA, allowing for the selection of cells in which theexogenous DNA has integrated into the host cell genome. Theidentification of the targeting event may also be facilitated by the useof one or more marker genes exhibiting the property of negativeselection, such that the negatively selectable marker is linked to theexogenous DNA, but configured such that the negatively selectable markerflanks the targeting sequence, and such that a correct homologousrecombination event with sequences in the host cell genome does notresult in the stable integration of the negatively selectable marker.Markers useful for this purpose include the Herpes Simplex Virusthymidine kinase (TK) gene or the bacterial xanthine-guaninephosphoribosyl-transferase (gpt) gene.

[0138] The gene targeting or gene activation techniques which can beused in accordance with this aspect of the invention are moreparticularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat.No. 5,578,461 to Sherwin et al.; International Application No.PCT/US92/09627 (WO93/09222) by Selden et al.; and InternationalApplication No. PCT/US90/06436 (WO91/06667) by Skoultchi et al., each ofwhich is incorporated by reference herein in its entirety.

[0139] In general, techniques for preparing polyclonal and monoclonalantibodies as well as hybridomas capable of producing the desiredantibody are well known in the art (Campbell, A. M., MonoclonalAntibodies Technology: Laboratory Techniques in Biochemistry andMolecular Biology, Elsevier Science Publishers, Amsterdam, TheNetherlands (1984); St. Groth et al., J. Immunol. 35:1-21 (1990); Kohlerand Milstein, Nature 256:495-497 (1975)), the trioma technique, thehuman B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72(1983); Cole et al., in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc. (1985), pp. 77-96).

[0140] Methods for immunization are well known in the art. Such methodsinclude subcutaneous or interperitoneal injection of the polypeptide.One skilled in the art will recognize that the amount of the proteinencoded by the reporter gene of the present invention used forimmunization will vary based on the animal which is immunized, theantigenicity of the peptide and the site of injection.

[0141] The polypeptide or protein of the invention which is used as animmunogen may be modified or administered in an adjuvant in order toincrease the polypeptide or protein's antigenicity. Methods ofincreasing the antigenicity of a polypeptide or protein are well knownin the art and include, but are not limited to, coupling the antigenwith a heterologous protein (such as globulin or β-galactosidase) orthrough the inclusion of an adjuvant during immunization.

[0142] For monoclonal antibodies, spleen cells from the immunizedanimals are removed, fused with myeloma cells, such as SP2/0-Ag14myeloma cells, and allowed to become monoclonal antibody producinghybridoma cells.

[0143] Any one of a number of methods well known in the art can be usedto identify the hybridoma cell which produces an antibody with thedesired characteristics. These include screening the hybridomas with anELISA assay, western blot analysis, or radioimmunoassay (Lutz et al.,Exp. Cell Research. 175:109-124 (1988)).

[0144] Hybridomas secreting the desired antibodies are cloned and theclass and subclass is determined using procedures known in the art(Campbell, A. M., Monoclonal Antibody Technology: Laboratory Techniquesin Biochemistry and Molecular Biology, Elsevier Science Publishers,Amsterdam, The Netherlands (1984)).

[0145] For polyclonal antibodies, antibody containing antisera isisolated from the immunized animal and is screened for the presence ofantibodies with the desired specificity using one of the above-describedprocedures.

[0146] The present invention further provides the above-describedantibodies in detectably labeled form. Antibodies can be detectablylabeled through the use of radioisotopes, affinity labels (such asbiotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase,alkaline phosphatase, etc.) fluorescent labels (such as FITC orrhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishingsuch labeling are well-known in the art, for example, see (Sternberger,L. A. et al., J. Histochem. CytoChem. 18:315 (1970); Bayer, E. A. etal., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129(1972); Goding, J. W. J. Immunol. Meth. 13:215 (1976)).

[0147] The labeled antibodies of the present invention can be used forin vitro, in vivo, and in situ assays to identify cells or tissues inwhich the polypeptide or protein of the invention is expressed.

[0148] The present invention further provides the above-describedantibodies immobilized on a solid support. Examples of such solidsupports include plastics such as polycarbonate, complex carbohydratessuch as agarose and sepharose, acrylic resins and such as polyacrylamideand latex beads. Techniques for coupling antibodies to such solidsupports are well known in the art (Weir, D. M. et al., ‘Handbook ofExperimental Immunology’ 4th Ed., Blackwell Scientific Publications,Oxford, England, Chapter 10 (1986); Jacoby, W. D. et al., Meth. Enzym.34 Academic Press, N.Y. (1974)). The immobilized antibodies of thepresent invention can be used for immuno-affinity purification of hostcells that are expressing the polypeptide or protein of the invention.

[0149] Host cells are transfected or preferably infected or transformedwith the above-described vectors, and cultured in nutrient mediaappropriate for selecting transductants or transformants containing thevector.

[0150] The host cells which express the polypeptide or protein of theinvention product may be identified by at least four general approaches;(a) DNA-DNA or DNA-RNA hybridization; (b) the presence or absence ofgene functions; (c) assessing the level of transcription as measured bythe expression of mRNA transcripts in the host cell; and (d) detectionof the gene product as measured by immunoassay or by its biologicalactivity.

[0151] In the first approach, the presence of the polypeptide or proteinof the invention inserted in the vector can be detected by DNA-DNA orDNA-RNA hybridization using probes comprising nucleotide sequences thatare homologous to the polypeptide or protein of the invention,respectively, or portions or derivatives thereof.

[0152] In the second approach, the recombinant expression vector/hostsystem can be identified and selected based upon the presence or absenceof certain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.). For example,if the polypeptide or protein of the invention is inserted within amarker gene sequence of the vector, recombinant cells containing thepolypeptide or protein of the invention can be identified by the absenceof the marker gene function. Alternatively, a marker gene can be placedin tandem with the polypeptide or protein of the invention under thecontrol of the same or different promoter used to control the expressionof the polypeptide or protein of the invention. Expression of the markerin response to induction or selection indicates expression of thepolypeptide or protein of the invention.

[0153] In the third approach, transcriptional activity of thepolypeptide or protein of the invention can be assessed by hybridizationassays. For example, RNA can be isolated and analyzed by Northern blotusing a probe homologous to the polypeptide or protein of the inventionor particular portions thereof. Alternatively, total nucleic acids ofthe host cell may be extracted and assayed for hybridization to suchprobes.

[0154] In the fourth approach, the expression of a product from thepolypeptide or protein of the invention can be assessed immunologically,for example by Western blots, immunoassays such asradioimmuno-precipitation, enzyme-linked immunoassays and the like.

[0155] The polynucleotides of the invention also provide polynucleotidesincluding nucleotide sequences that are substantially equivalent to thepolynucleotides of the invention. Polynucleotides according to theinvention can have at least about 80%, more typically at least about90%, and even more typically at least about 95%, sequence identity to apolynucleotide of the invention. The invention also provides thecomplement of the polynucleotides including a nucleotide sequence thathas at least about 80%, more typically at least about 90%, and even moretypically at least about 95%, sequence identity to a polynucleotideencoding a polypeptide recited above. The polynucleotide can be DNA(genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithmsfor obtaining such polynucleotides are well known to those of skill inthe art and can include, for example, methods for determininghybridization conditions which can routinely isolate polynucleotides ofthe desired sequence identities.

[0156] A polynucleotide according to the invention can be joined to anyof a variety of other nucleotide sequences by well-establishedrecombinant DNA techniques (see Sambrook J et al. (1989) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY). Usefulnucleotide sequences for joining to polypeptides include an assortmentof vectors, e.g., plasmids, cosmids, lambda phage derivatives,phagemids, and the like, that are well known in the art. Accordingly,the invention also provides a vector including a polynucleotide of theinvention and a host cell containing the polynucleotide. In general, thevector contains an origin of replication functional in at least oneorganism, convenient restriction endonuclease sites, and a selectablemarker for the host cell. Vectors according to the invention includeexpression vectors, replication vectors, probe generation vectors, andsequencing vectors. A host cell according to the invention can be aprokaryotic or eukaryotic cell and can be a unicellular organism or partof a multicellular organism.

[0157] The sequences falling within the scope of the present inventionare not limited to the specific sequences herein described, but alsoinclude a representative fragment thereof, or a nucleotide sequence atleast 99.9% identical to a nucleic acid of the invention. Furthermore,to accommodate codon variability, the invention includes nucleic acidmolecules encoding the polypeptide sequences of the invention. In otherwords, in the coding region of a polypeptide sequence of the invention,substitution of one codon for another which encodes the same amino acidis expressly contemplated. Any specific sequence disclosed herein can bereadily screened for errors by resequencing a particular fragment, suchas an ORF, in both directions (i.e., sequence both strands).

[0158] The present invention further provides recombinant constructscomprising a nucleic acid of the invention, or a fragment thereof. Therecombinant constructs of the present invention comprise a vector, suchas a plasmid or viral vector, into which a nucleic acid of theinvention, or a fragment thereof is inserted, in a forward or reverseorientation. In the case of a vector comprising one of the ORFs of thepresent invention, the vector may further comprise regulatory sequences,including for example, a promoter, operably linked to the ORF. Forvectors comprising the EMFs and UMFs of the present invention, thevector may further comprise a marker sequence or heterologous ORFoperably linked to the EMF or UMF. Large numbers of suitable vectors andpromoters are known to those of skill in the art and are commerciallyavailable for generating the recombinant constructs of the presentinvention. The following vectors are provided by way of example.Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a,pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3,pDR540, pRITS (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).

[0159] The isolated polynucleotide of the invention may be operablylinked to an expression control sequence such as the pMT2 or pEDexpression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19,4485-4490 (1991), in order to produce the protein or polypeptiderecombinantly. Many suitable expression control sequences are known inthe art. General methods of expressing recombinant proteins are alsoknown and are exemplified in R. Kaufman, Methods in Enzymology 185,537-566 (1990). As defined herein “operably linked” means that theisolated polynucleotide of the invention and an expression controlsequence are situated within a vector or cell in such a way that theprotein or polypeptide is expressed by a host cell which has beentransformed (transfected) with the ligated polynucleotide/expressioncontrol sequence.

[0160] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), andtrc. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art. Generally,recombinant expression vectors will include origins of replication andselectable markers permitting transformation of the host cell, e.g., theampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and apromoter derived from a highly-expressed gene to direct transcription ofa downstream structural sequence. Such promoters can be derived fromoperons encoding glycolytic enzymes such as 3-phosphoglycerate kinase(PGK), a-factor, acid phosphatase, or heat shock proteins, among others.The heterologous structural sequence is assembled in appropriate phasewith translation initiation and termination sequences, and preferably, aleader sequence capable of directing secretion of translated protein orpolypeptide into the periplasmic space or extracellular medium.Optionally, the heterologous sequence can encode a fusion proteinincluding an N-terminal identification peptide imparting desiredcharacteristics, e.g., stabilization or simplified purification ofexpressed recombinant product. Useful expression vectors for bacterialuse are constructed by inserting a structural DNA sequence encoding adesired protein or polypeptide together with suitable translationinitiation and termination signals in operable reading phase with afunctional promoter. The vector will comprise one or more phenotypicselectable markers and an origin of replication to ensure maintenance ofthe vector and to, if desirable, provide amplification within the host.Suitable prokaryotic hosts for transformation include E. coli, Bacillussubtilis, Salmonella typhimurium and various species within the generaPseudomonas, Streptomyces, and Staphylococcus, although others may alsobe employed as a matter of choice.

[0161] As a representative but non-limiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec,Madison, Wis., USA). These pBR322 ‘backbone’ sections are combined withan appropriate promoter and the structural sequence to be expressed.Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced or derepressed by appropriate means (e.g., temperature shift orchemical induction) and cells are cultured for an additional period.Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

[0162] Included within the scope of the nucleic acid sequences of theinvention are nucleic acid sequences that hybridize under stringentconditions to a polynucleotide of the invention, which polynucleotide isgreater than about 10 bp, preferably 20-50 bp, and even greater than 100bp. In accordance with the invention, polynucleotide sequences of theinvention, or functional equivalents thereof, may be used to generaterecombinant DNA molecules that direct the expression of thatpolynucleotide, or a functional equivalent thereof, in appropriate hostcells.

[0163] The polynucleotides of the invention are further directed tosequences which encode variants of the polypeptides or proteins of theinvention. These amino acid sequence variants may be prepared by methodsknown in the art by introducing appropriate nucleotide changes into anative or variant polynucleotide. There are two variables in theconstruction of amino acid sequence variants: the location of themutation and the nature of the mutation. The amino acid sequencevariants of the nucleic acids are preferably constructed by mutating thepolynucleotide to give an amino acid sequence that does not occur innature. These amino acid alterations can be made at sites that differ inthe nucleic acids from different species (variable positions) or inhighly conserved regions (constant regions). Sites at such locationswill typically be modified in series, e.g., by substituting first withconservative choices (e.g., hydrophobic amino acid to a differenthydrophobic amino acid) and then with more distant choices (e.g.,hydrophobic amino acid to a charged amino acid), and then deletions orinsertions may be made at the target site. Amino acid sequence deletionsgenerally range from about 1 to 30 residues, preferably about 1 to 10residues, and are typically contiguous. Amino acid insertions includeamino- and/or carboxyl-terminal fusions ranging in length from one toone hundred or more residues, as well as intrasequence insertions ofsingle or multiple amino acid residues. Intrasequence insertions mayrange generally from about 1 to 10 amino residues, preferably from 1 to5 residues. Examples of terminal insertions include the heterologoussignal sequences necessary for secretion or for intracellular targetingin different host cells.

[0164] Amino acid sequence deletions generally range from about 1 to 30residues, preferably about 1 to 10 residues, and are typicallycontiguous. Amino acid insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one to one hundred ormore residues, as well as intrasequence insertions of single or multipleamino acid residues. Intrasequence insertions may range generally fromabout 1 to 10 amino residues, preferably from 1 to 5 residues. Examplesof terminal insertions include the heterologous signal sequencesnecessary for secretion or for intracellular targeting in different hostcells.

[0165] PCR may also be used to create amino acid sequence variants ofthe polynucleotides of the invention. When small amounts of template DNAare used as starting material, primer(s) that differs slightly insequence from the corresponding region in the template DNA can generatethe desired amino acid variant. PCR amplification results in apopulation of product DNA fragments that differ from the polynucleotidetemplate encoding the polypeptide or protein at the position specifiedby the primer. The product DNA fragments replace the correspondingregion in the plasmid and this gives the desired amino acid variant.

[0166] In a preferred method, polynucleotides encoding thepolynucleotides of the invention are changed via site-directedmutagenesis. This method uses oligonucleotide sequences that encode thepolynucleotide sequence of the desired amino acid variant, as well as asufficient adjacent nucleotide on both sides of the changed amino acidto form a stable duplex on either side of the site of being changed. Ingeneral, the techniques of site-directed mutagenesis are well known tothose of skill in the art and this technique is exemplified bypublications such as, Edelman et al., DNA 2:183 (1983). A versatile andefficient method for producing site-specific changes in a polynucleotidesequence was published by Zoller and Smith, Nucleic Acids Res.10:6487-6500 (1982). PCR may also be used to create amino acid sequencevariants of the novel nucleic acids. When small amounts of template DNAare used as starting material, primer(s) that differs slightly insequence from the corresponding region in the template DNA can generatethe desired amino acid variant. PCR amplification results in apopulation of product DNA fragments that differ from the polynucleotidetemplate encoding the polypeptide at the position specified by theprimer. The product DNA fragments replace the corresponding region inthe plasmid and this gives the desired amino acid variant.

[0167] A further technique for generating amino acid variants is thecassette mutagenesis technique described in Wells et al., Gene 34:315(1985); and other mutagenesis techniques well known in the art, such as,for example, the techniques in Sambrook et al., supra, and CurrentProtocols in Molecular Biology, Ausubel et al. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be used in the practice of the invention for the cloning andexpression of these novel nucleic acids. Such DNA sequences includethose which are capable of hybridizing to the appropriate novel nucleicacid sequence under stringent conditions.

[0168] The invention encompasses polypeptides or proteins encoded by thepolynucleotides of the invention. Fragments of the polypeptides orproteins of the present invention which are capable of exhibitingbiological activity are also encompassed by the present invention.Fragments of the protein or polypeptide may be in linear form or theymay be cyclized using known methods, for example, as described in H. U.Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S.McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both ofwhich are incorporated herein by reference.

[0169] The present invention also provides both full-length and matureforms of the polypeptides or proteins of the invention. The full-lengthform of the such polypeptides or proteins can be identified bytranslation of the nucleotide sequence of each polynucleotide of theinvention. The mature form of such polypeptide or protein may beobtained by expression of the full-length polynucleotide in a suitablemammalian cell or other host cell. The sequence of the mature form ofthe polypeptide or protein may also be determinable from the amino acidsequence of the full-length form.

[0170] Where the protein or polypeptide of the present invention ismembrane-bound (e.g., is a receptor), the present invention alsoprovides for soluble forms of such protein or polypeptide. In such formspart or all of the intracellular and transmembrane domains of theprotein or polypeptide are deleted such that the protein or polypeptideis fully secreted from the cell in which it is expressed. Theintracellular and transmembrane domains of proteins or polypeptides ofthe invention can be identified in accordance with known techniques fordetermination of such domains from sequence information.

[0171] The invention also relates to methods for producing a polypeptideor protein of the invention comprising growing a culture of the cells ofthe invention in a suitable culture medium, and purifying the protein orpolypeptide of the invention from the culture. For example, the methodsof the invention include a process for producing a polypeptide orprotein of the invention in which a host cell containing a suitableexpression vector that includes a polynucleotide or protein of theinvention is cultured under conditions that allow expression of theencoded polypeptide or protein The polypeptide or protein can berecovered from the culture, conveniently from the culture medium, andfurther purified.

[0172] The invention further provides a polypeptide or protein of theinvention including an amino acid sequence that is substantiallyequivalent to an amino acid sequence encoded by a polynucleotide of theinvention. Polypeptides or proteins according to the invention can haveat least about 95%, and more typically at least about 98%, sequenceidentity to an amino acid sequence encoded by a polynucleotide of theinvention.

[0173] The present invention further provides isolated polypeptides orproteins encoded by the polyncueltides of the present invention or bydegenerate variants of the polynucleotides of the present invention. By‘degenerate variant’ is intended polynucleotides which differ from anucleic acid fragment of the present invention (e.g., an ORF) bynucleotide sequence but, due to the degeneracy of the genetic code,encode an identical polypeptide sequence. Preferred polynucleotides ofthe present invention are the ORFs that encode proteins or polypeptides.A variety of methodologies known in the art can be utilized to obtainany one of the isolated polypeptides or proteins of the presentinvention. At the simplest level, the amino acid sequence can besynthesized using commercially available peptide synthesizers. This isparticularly useful in producing small peptides and fragments of largerpolypeptides. Fragments are useful, for example, in generatingantibodies against the native polypeptide. In an alternative method, thepolypeptide or protein is purified from bacterial cells which naturallyproduce the polypeptide or protein. One skilled in the art can readilyfollow known methods for isolating polypeptides and proteins in order toobtain one of the isolated polypeptides or proteins of the presentinvention. These include, but are not limited to, immunochromatography,HPLC, size-exclusion chromatography, ion-exchange chromatography, andimmuno-affinity chromatography. See, e.g., Scopes, Protein Purification:Principles and Practice, Springer-Verlag (1994); Sambrook, et al., inMolecular Cloning: A Laboratory Manual; Ausubel et al., CurrentProtocols in Molecular Biology.

[0174] The polypeptides and proteins of the present invention canalternatively be purified from cells which have been altered to expressthe desired polypeptide or protein. As used herein, a cell is said to bealtered to express a desired polypeptide or protein when the cell,through genetic manipulation, is made to produce a polypeptide orprotein which it normally does not produce or which the cell normallyproduces at a lower level. One skilled in the art can readily adaptprocedures for introducing and expressing either recombinant orsynthetic sequences into eukaryotic or prokaryotic cells in order togenerate a cell which produces one of the polypeptides or proteins ofthe present invention. The purified polypeptides or proteins can be usedin in vitro binding assays which are well known in the art to identifymolecules which bind to the polypeptides or proteins. These moleculesinclude but are not limited to, for e.g., small molecules, moleculesfrom combinatorial libraries, antibodies or other proteins orpolypeptides. The molecules identified in the binding assay are thentested for antagonist or agonist activity in in vivo tissue culture oranimal models that are well known in the art. In brief, the moleculesare titrated into a plurality of cell cultures or animals and thentested for either cell/animal death or prolonged survival of theanimal/cells.

[0175] In addition, the binding molecules may be complexed with toxins,e.g., ricin or cholera, or with other compounds that are toxic to cells.The toxin-binding molecule complex is then targeted to the tumor orother cell by the specificity of the binding molecule.

[0176] The protein or polypeptide of the invention may also be expressedas a product of transgenic animals, e.g., as a component of the milk oftransgenic cows, goats, pigs, or sheep which are characterized bysomatic or germ cells containing a polynucleotide encoding the proteinor polypeptide of the invention.

[0177] The protein or polypeptide of the invention may also be producedby known conventional chemical synthesis. Methods for constructing theproteins or polypeptides of the present invention by synthetic means areknown to those skilled in the art. The synthetically-constructedproteins or polypeptides, by virtue of sharing primary, secondary ortertiary structural and/or conformational characteristics with proteinsor polypeptides may possess biological properties in common therewith,including protein activity. Thus, they may be employed as biologicallyactive or immunological substitutes for natural, purified proteins orpolypeptides of the invention in screening of therapeutic compounds andin immunological processes for the development of antibodies.

[0178] The proteins or polypeptides of the invention provided hereinalso include proteins or polypeptides characterized by amino acidsequences similar to those of purified proteins or polypeptides of theinvention but into which modifications are naturally provided ordeliberately engineered. For example, modifications in the peptide orDNA sequences can be made by those skilled in the art using knowntechniques. Modifications of interest in the protein sequences mayinclude the alteration, substitution, replacement, insertion or deletionof a selected amino acid residue in the coding sequence. For example,one or more of the cysteine residues may be deleted or replaced withanother amino acid to alter the conformation of the molecule. Techniquesfor such alteration, substitution, replacement, insertion or deletionare well known to those skilled in the art (see, e.g., U.S. Pat. No.4,518,584). Preferably, such alteration, substitution, replacement,insertion or deletion retains the desired activity of the protein orpolypeptide.

[0179] Other fragments and derivatives of the polypeptides or proteinsof the invention which would be expected to retain protein activity inwhole or in part and may thus be useful for screening or otherimmunological methodologies may also be easily made by those skilled inthe art given the disclosures herein. Such modifications are encompassedby the present invention.

[0180] The protein or polypeptide of the invention may also be producedby operably linking a polynucleotide of the invention to suitablecontrol sequences in one or more insect expression vectors, andemploying an insect expression system. Materials and methods forbaculovirus/insect cell expression systems are commercially available inkit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (theMaxBat.RTM. kit), and such methods are well known in the art, asdescribed in Summers and Smith, Texas Agricultural Experiment StationBulletin No. 1555 (1987), incorporated herein by reference. As usedherein, an insect cell capable of expressing a polynucleotide of thepresent invention is “transformed.”

[0181] The protein or polypeptide of the invention may be prepared byculturing transformed host cells under culture conditions suitable toexpress the recombinant protein or polypeptide. The resulting expressedprotein or polypeptide may then be purified from such culture (i.e.,from culture medium or cell extracts) using known purificationprocesses, such as gel filtration and ion exchange chromatography. Thepurification of the protein or polypeptide may also include an affinitycolumn containing agents which will bind to the protein or polypeptide;one or more column steps over such affinity resins as concanavalinA-agarose, heparin-toyopearl.RTM. or Cibacrom blue 3GA Sepharose.RTM.;one or more steps involving hydrophobic interaction chromatography usingsuch resins as phenyl ether, butyl ether, or propyl ether; orimmunoaffinity chromatography.

[0182] Alternatively, the protein or polypeptide of the invention mayalso be expressed in a form which will facilitate purification. Forexample, it may be expressed as a fusion protein, such as those ofmaltose binding protein (MBP), glutathione-S-transferase (GST) orthioredoxin (TRX). Kits for expression and purification of such fusionproteins are commercially available from New England BioLab (Beverly,Mass.), Pharmacia (Piscataway, N.J.) and In Vitrogen, respectively. Theprotein or polypeptide can also be tagged with an epitope andsubsequently purified by using a specific antibody directed to suchepitope. One such epitope (“Flag”) is commercially available from Kodak(New Haven, Conn.).

[0183] Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the protein or polypeptide. Some or all ofthe foregoing purification steps, in various combinations, can also beemployed to provide a substantially homogeneous isolated recombinantprotein or polypeptide. The protein or polypeptide thus purified issubstantially free of other mammalian proteins and is defined inaccordance with the present invention as an “isolated protein.”

[0184] The polynucleotides and polypeptides of the present invention areexpected to exhibit one or more of the uses or biological activities(including those associated with assays cited herein) identified below.Uses or activities described for the polypeptides or proteins of thepresent invention may be provided by administration or use of suchproteins or polypeptides or by administration or use of polynucleotidesencoding such proteins or polypeptides (such as, for example, in genetherapies or vectors suitable for introduction of DNA).

[0185] The polynucleotides provided by the present invention can be usedby the research community for various purposes. The polynucleotides canbe used to express recombinant protein or polypeptide for analysis,characterization, diagnostic or therapeutic use; as markers for tissuesin which the target protein is abnormally or normally expressed (e.g.,constitutively or at a particular stage of tissue differentiation ordevelopment or in disease states); as molecular weight markers onSouthern gels; as chromosome markers or tags (when labeled) to identifychromosomes or to map related gene positions; to compare with endogenousDNA sequences in patients to identify potential genetic disorders; asprobes to hybridize and thus discover novel, related DNA sequences; as asource of information to derive PCR primers for genetic fingerprinting;as a probe to “subtract-out” known sequences in the process ofdiscovering other novel polynucleotides; for selecting and makingoligomers for attachment to a “gene chip” or other support, includingfor examination of expression patterns; for attachment to a substrate tomake an antibody chip for examining protein (target) expression patternsor target expression levels or the presence of the target, and as anantigen to raise anti-idiotype antibodies. When the target protein bindsor potentially binds to another protein or other factor, thepolynucleotides of the invention can also be used in interaction trapassays (such as, for example, that described in Gyuris et al., Cell75:791-803 (1993)) to identify polynucleotides encoding the otherprotein or factor with which binding occurs or to identify other factorsor proteins involved in the binding interation.

[0186] The proteins or polypeptides provided by the present inventioncan similarly be used to determine biological activity, including in apanel of multiple proteins or polypeptides for high-throughputscreening; as a reagent (including the labeled reagent) in assaysdesigned to quantitatively determine levels of the target protein inbiological samples; as markers for tissues in which the target proteinof the invention is normally or abnormally expressed (eitherconstitutively or at a particular stage of tissue differentiation ordevelopment or in a disease state); and, of course, to isolatecorrelative receptors or ligands. Where the target protein binds orpotentially binds to another protein or factor (such as, for example, ina receptor-ligand interaction), the polypeptide of the invention can beused to identify the other protein or factor with which binding occursor to identify inhibitors of the binding interaction. Proteins involvedin these binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction.

[0187] Any or all of these research utilities are capable of beingdeveloped into reagent grade or kit format for commercialization asresearch products.

[0188] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods includewithout limitation “Molecular Cloning: A Laboratory Manual”, 2d ed.,Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T.Maniatis eds., 1989, and “Methods in Enzymology: Guide to MolecularCloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmeleds., 1987.

[0189] Polynucleotides, proteins, and polypeptides of the presentinvention can also be used as nutritional sources or supplements. Suchuses include without limitation use as a protein or polypeptide or aminoacid supplement, use as a carbon source, use as a nitrogen source anduse as a source of carbohydrate. In such cases the protein, polypeptide,or polynucleotide of the invention can be added to the feed of aparticular organism or can be administered as a separate solid or liquidpreparation, such as in the form of powder, pills, solutions,suspensions or capsules. In the case of microorganisms, the protein,polypeptide, or polynucleotide of the invention can be added to themedium in or on which the microorganism is cultured.

[0190] A protein or polypeptide of the present invention may exhibitcytokine, cell proliferation (either inducing or inhibiting) or celldifferentiation (either inducing or inhibiting) activity or may induceproduction of other cytokines in certain cell populations, or may be anantogonist or agonist of any of the above. A polynucleotide of theinvention can encode a polypeptide exhibiting such attributes. Manyprotein factors discovered to date, including all known cytokines, haveexhibited activity in one or more factor-dependent cell proliferationassays, and hence the assays serve as a convenient confirmation ofcytokine agonist or antagonist activity. The activity of a protein orpolypeptide of the present invention is evidenced by any one of a numberof routine factor dependent cell proliferation assays for cell linesincluding, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3,MC9/G, M+ (preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7eand CMK.

[0191] The activity of a protein or polypeptide of the invention may,among other means, be measured by the following methods:

[0192] Assays for T-cell or thymocyte proliferation include withoutlimitation those described in: Current Protocols in Immunology, Ed by J.E. coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, Invitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7,Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolliet al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I.Immunol. 149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761,1994.

[0193] Assays for cytokine production and/or proliferation of spleencells, lymph node cells or thymocytes include, without limitation, thosedescribed in: Polyclonal T cell stimulation, Kruisbeek, A. M. andShevach, E. M. In Current Protocols in Immunology. J. E. e.a. Coliganeds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; andMeasurement of mouse and human interleukin gamma., Schreiber, R. D. InCurrent Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp.6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.

[0194] Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described in:Measurement of Human and Murine Interleukin 2 and Interleukin 4,Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols inImmunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wileyand Sons, Toronto. 1991; devries et al., J. Exp. Med. 173:1205-1211,1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse andhuman interleukin 6—Nordan, R. In Current Protocols in Immunology. J. E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto.1991; Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861, 1986;Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark,S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. e.a.Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;Measurement of mouse and human Interleukin 9—Ciarletta, A., Giannotti,J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology.J. E. e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto.1991.

[0195] Assays for T-cell clone responses to antigens (which willidentify, among others, proteins that affect APC-T cell interactions aswell as direct T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, In vitro assays for Mouse LymphocyteFunction; Chapter 6, Cytokines and their cellular receptors; Chapter 7,Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad.Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun.11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takaiet al., J. Immunol. 140:508-512, 1988.

[0196] In all the above assays, the polypeptide or protein of theinvention is added into the assay system and activity of a targetcytokine is determined in the presence and absence of the polypeptide orprotein of the invention.

[0197] Further, the polypeptides of the ivention may be used to examinethe expression level or presence of a cytokine. In alternateembodiments, the detection of a cytokine or of a the level of a cytokinewill be diagnostic for a disease state or condition.

[0198] A protein or polypeptide of the present invention may alsoexhibit immune stimulating or immune suppressing activity, or may beantagonists or agonists of either activity, including without limitationthe activities for which assays are described herein. A polynucleotideof the invention can encode a polypeptide or protein exhibiting suchactivities. A protein or polypeptide of the invention may be useful inthe treatment and/or detection (e.g., a diagnostic) of various immunedeficiencies and disorders (including severe combined immunodeficiency(SCID)), e.g., in regulating (up or down) growth and proliferation of Tand/or B lymphocytes, as well as effecting the cytolytic activity of NKcells and other cell populations. These immune deficiencies may begenetic or be caused by viral (e.g., HIV) as well as bacterial or fungalinfections, or may result from autoimmune disorders. More specifically,infectious diseases caused by viral, bacterial, fungal or otherinfections may be treatable or detectable (e.g., a diagnostic test)using a protein or polypeptide of the present invention, includinginfections by HIV, hepatitis viruses, herpesviruses, mycobacteria,Leishmania spp., malaria spp. and various fungal infections such ascandidiasis. Of course, in this regard, a protein or polypeptide of thepresent invention may also be useful where a boost to the immune systemgenerally may be desirable, i.e., in the treatment of cancer.

[0199] Autoimmune disorders which may be treated or detected using aprotein or polypeptide of the present invention include, for example,connective tissue disease, multiple sclerosis, systemic lupuserythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependentdiabetes mellitis, myasthenia gravis, graft-versus-host disease andautoimmune inflammatory eye disease. Such a protein or polypeptide ofthe present invention may also to be useful in the treatment of allergicreactions and conditions, such as asthma (particularly allergic asthma)or other respiratory problems. Other conditions, in which immunesuppression is desired (including, for example, organ transplantation),may also be treatable using a protein or polypeptide of the presentinvention.

[0200] Using the proteins or polypeptides of the invention it may alsobe possible to modulate immune responses, in a number of ways. Downregulation may be in the form of inhibiting or blocking an immuneresponse already in progress or may involve preventing the induction ofan immune response. The functions of activated T cells may be inhibitedby suppressing T cell responses or by inducing specific tolerance in Tcells, or both. Immunosuppression of T cell responses is generally anactive, non-antigen-specific, process which requires continuous exposureof the T cells to the suppressive agent. Tolerance, which involvesinducing non-responsiveness or anergy in T cells, is distinguishablefrom immunosuppression in that it is generally antigen-specific andpersists after exposure to the tolerizing agent has ceased.Operationally, tolerance can be demonstrated by the lack of a T cellresponse upon reexposure to specific antigen in the absence of thetolerizing agent.

[0201] Down regulating or preventing one or more antigen functions(including without limitation B lymphocyte antigen functions (such as,for example, B7)), e.g., preventing high level lymphokine synthesis byactivated T cells, will be useful in situations of tissue, skin andorgan transplantation and in graft-versus-host disease (GVHD). Forexample, blockage of T cell function should result in reduced tissuedestruction in tissue transplantation. Typically, in tissue transplants,rejection of the transplant is initiated through its recognition asforeign by T cells, followed by an immune reaction that destroys thetransplant. The administration of a molecule which inhibits or blocksinteraction of a B7 lymphocyte antigen with its natural ligand(s) onimmune cells (such as a soluble, monomeric form of a peptide having B7-2activity alone or in conjunction with a monomeric form of a peptidehaving an activity of another B lymphocyte antigen (e.g., B7-1, B7-3) orblocking antibody), prior to transplantation can lead to the binding ofthe molecule to the natural ligand(s) on the immune cells withouttransmitting the corresponding costimulatory signal. Blocking Blymphocyte antigen function in this matter prevents cytokine synthesisby immune cells, such as T cells, and thus acts as an immunosuppressant.Moreover, the lack of costimulation may also be sufficient to anergizethe T cells, thereby inducing tolerance in a subject. Induction oflong-term tolerance by B lymphocyte antigen-blocking reagents may avoidthe necessity of repeated administration of these blocking reagents. Toachieve sufficient immunosuppression or tolerance in a subject, it mayalso be necessary to block the function of a combination of B lymphocyteantigens.

[0202] The efficacy of particular blocking reagents in preventing organtransplant rejection or GVHD can be assessed using animal models thatare predictive of efficacy in humans. Examples of appropriate systemswhich can be used include allogeneic cardiac grafts in rats andxenogeneic pancreatic islet cell grafts in mice, both of which have beenused to examine the immunosuppressive effects of CTLA4Ig fusion proteinsin vivo as described in Lenschow et al., Science 257:789-792 (1992) andTurka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). Inaddition, murine models of GVHD (see Paul ed., Fundamental Immunology,Raven Press, New York, 1989, pp. 846-847) can be used to determine theeffect of blocking B lymphocyte antigen function in vivo on thedevelopment of that disease. Further, the polypeptides of the inventioncan be used to detect GVHD after organ transplant.

[0203] Blocking antigen function may also be therapeutically useful fortreating autoimmune diseases. Many autoimmune disorders are the resultof inappropriate activation of T cells that are reactive against selftissue and which promote the production of cytokines and autoantibodiesinvolved in the pathology of the diseases. Preventing the activation ofautoreactive T cells may reduce or eliminate disease symptoms.Administration of reagents which block costimulation of T cells bydisrupting receptor:ligand interactions of B lymphocyte antigens can beused to inhibit T cell activation and prevent production ofautoantibodies or T cell-derived cytokines which may be involved in thedisease process. Additionally, blocking reagents may induceantigen-specific tolerance of autoreactive T cells which could lead tolong-term relief from the disease. The efficacy of blocking reagents inpreventing or alleviating autoimmune disorders can be determined using anumber of well-characterized animal models of human autoimmune diseases.Examples include murine experimental autoimmune encephalitis, systemiclupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murineautoimmune collagen arthritis, diabetes mellitus in NOD mice and BBrats, and murine experimental myasthenia gravis (see Paul ed.,Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).Further, polypeptides of the invention can be used to diagnose an immunedisorder and/or the susceptibility of an organism for an immunedisorder.

[0204] Upregulation of an antigen function (preferably a B lymphocyteantigen function), as a means of up regulating immune responses, mayalso be useful in therapy. Upregulation of immune responses may be inthe form of enhancing an existing immune response or eliciting aninitial immune response. For example, enhancing an immune responsethrough stimulating B lymphocyte antigen function may be useful in casesof viral infection. In addition, systemic viral diseases such asinfluenza, the common cold, and encephalitis might be alleviated by theadministration of stimulatory forms of B lymphocyte antigenssystemically.

[0205] Alternatively, anti-vital immune responses may be enhanced in aninfected patient by removing T cells from the patient, costimulating theT cells in vitro with viral antigen-pulsed APCs either expressing apeptide of the present invention or together with a stimulatory form ofa soluble peptide of the present invention and reintroducing the invitro activated T cells into the patient. Another method of enhancinganti-viral immune responses would be to isolate infected cells from apatient, transfect them with a nucleic acid encoding a protein orpolypeptide of the present invention as described herein such that thecells express all or a portion of the protein or polypeptide on theirsurface, and reintroduce the transfected cells into the patient. Theinfected cells would now be capable of delivering a costimulatory signalto, and thereby activate, T cells in vivo.

[0206] The presence of a polypeptide or protein of the present inventionhaving the activity of a B lymphocyte antigen(s) on the surface of thetumor cell provides the necessary costimulation signal to T cells toinduce a T cell mediated immune response against the transfected tumorcells. In addition, tumor cells which lack MHC class I or MHC class IImolecules, or which fail to reexpress sufficient mounts of MHC class Ior MHC class II molecules, can be transfected with nucleic acid encodingall or a portion of (e.g., a cytoplasmic-domain truncated portion) of anMHC class I a chain protein and β₂ microglobulin protein or an MHC classII “chain protein and an MHC class II β chain protein to thereby expressMHC class I or MHC class II proteins on the cell surface. Expression ofthe appropriate class I or class II MHC in conjunction with a peptidehaving the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3)induces a T cell mediated immune response against the transfected tumorcell. optionally, a gene encoding an antisense construct which blocksexpression of an MHC class II associated protein, such as the invariantchain, can also be cotransfected with a DNA encoding a peptide havingthe activity of a B lymphocyte antigen to promote presentation of tumorassociated antigens and induce tumor specific immunity. Thus, theinduction of a T cell mediated immune response in a human subject may besufficient to overcome tumor-specific tolerance in the subject.

[0207] The activity of a protein or polypeptide of the invention may,among other means, be measured by the following methods:

[0208] Suitable assays for thymocyte or splenocyte cytotoxicity include,without limitation, those described in: Current Protocols in Immunology,Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience(Chapter 3, In vitro assays for Mouse Lymphocyte Function 3.1-3.19;Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985;Takai et al., I. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982;Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol.137:3494-3500, 1986; Bowman et al., J. Virology 61:1992-1998; Takai etal., J. Immunol. 140:508-512, 1988; Bertagnolli et al., CellularImmunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092,1994.

[0209] Assays for T-cell-dependent immunoglobulin responses and isotypeswitching (which will identify, among others, proteins that modulateT-cell dependent antibody responses and that affect Th1/Th2 profiles)include, without limitation, those described in: Maliszewski, J.Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitroantibody production, Mond, J. J. and Brunswick, M. In Current Protocolsin Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, JohnWiley and Sons, Toronto. 1994.

[0210] Mixed lymphocyte reaction (MLR) assays (which will identify,among others, proteins that generate predominantly Th1 and CTLresponses) include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, In vitro assays for Mouse LymphocyteFunction 3.13.19; Chapter 7, Immunologic studies in Humans); Takai etal., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

[0211] Dendritic cell-dependent assays (which will identify, amongothers, proteins expressed by dendritic cells that activate naiveT-cells) include, without limitation, those described in: Guery et al.,J. Immunol. 134:536-544, 1995; Inaba et al., Journal of ExperimentalMedicine 173:549-559, 1991; Macatonia et al., Journal of Immunology154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993;Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal ofExperimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal ofClinical Investigation 94:797-807, 1994; and Inaba et al., Journal ofExperimental Medicine 172:631-640, 1990.

[0212] Assays for lymphocyte survival/apoptosis (which will identify,among others, proteins that prevent apoptosis after superantigeninduction and proteins that regulate lymphocyte homeostasis) include,without limitation, those described in: Darzynkiewicz et al., Cytometry13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca etal., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243,1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai etal., Cytometry 14:891-897, 1993; Gorczyca et al., International Journalof Oncology 1:639-648, 1992.

[0213] Assays for proteins that influence early steps of T-cellcommitment and development include, without limitation, those describedin: Antica et al., Blood 84:111-117, 1994; Fine et al., CellularImmunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995;Toki et al., Proc. Nat. Acad. Sci. USA 88:7548-7551, 1991.

[0214] A protein or polypeptide of the present invention may be usefulin regulation of hematopoiesis (as an antagonist or agonist) and,consequently, in the treatment and/or detection (e.g., a diagnostic) ofmyeloid or lymphoid cell deficiencies. Even marginal biological activityin support of colony forming cells or of factor-dependent cell linesindicates involvement in regulating hematopoiesis, e.g., in supportingthe growth and proliferation of erythroid progenitor cells alone or incombination with other cytokines, thereby indicating utility, forexample, in treating and/or detecting (e.g., a diagnostic) variousanemias or for use in conjunction with irradiation/chemotherapy tostimulate the production of erythroid precursors and/or erythroid cells;in supporting the growth and proliferation of myeloid cells such asgranulocytes and monocytes/macrophages (i.e., traditional CSF activity),for example, in conjunction with chemotherapy to prevent or treatconsequent myelo-suppression; in supporting the growth and proliferationof megakaryocytes and consequently of platelets thereby allowingprevention or treatment of various platelet disorders such asthrombocytopenia, and generally for use in place of or complimentary toplatelet transfusions; and/or in supporting the growth and proliferationof hematopoietic stem cells which are capable of maturing to any and allof the abovementioned hematopoietic cells and therefore find therapeuticutility in various stem cell disorders (such as those usually treatedwith transplantation, including, without limitation, aplastic anemia andparoxysmal nocturnal hemoglobinuria), as well as in repopulating thestem cell compartment post irradiation/chemotherapy, either in-vivo orex-vivo (i.e., in conjunction with bone marrow transplantation or withperipheral progenitor cell transplantation (homologous or heterologous))as normal cells or genetically manipulated for gene therapy.

[0215] The activity of a protein or polypeptide of the invention may,among other means, be measured by the following methods:

[0216] Suitable assays for proliferation and differentiation of varioushematopoietic lines are cited above.

[0217] Assays for embryonic stem cell differentiation (which willidentify, among others, proteins that influence embryonicdifferentiation hematopoiesis) include, without limitation, thosedescribed in: Johansson et al. Cellular Biology 15:141-151, 1995; Kelleret al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan etal., Blood 81:2903-2915, 1993.

[0218] Assays for stem cell survival and differentiation (which willidentify, among others, proteins that regulate lympho-hematopoiesis)include, without limitation, those described in: Methylcellulose colonyforming assays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;Primitive hematopoietic colony forming cells with high proliferativepotential, McNiece, I. K. and Briddell, R. A. In Culture ofHematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., ExperimentalHematology 22:353-359, 1994; Cobblestone area forming cell assay,Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I. Freshney, etal. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long termbone marrow cultures in the presence of stromal cells, Spooncer, E.,Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y.1994; Long term culture initiating cell assay, Sutherland, H. J. InCulture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp.139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

[0219] A protein or polypeptide of the present invention also may haveutility in compositions used for bone, cartilage, tendon, ligamentand/or nerve tissue growth or regeneration, as well as for wound healingand tissue repair and replacement, and in the treatment of burns,incisions and ulcers (as an antagonist or agonist).

[0220] A protein or polypeptide of the present invention, which acts asan antagonist or agonist of cartilage and/or bone growth, hasapplication in the healing of bone fractures and cartilage damage ordefects in humans and other animals. Such a preparation employing aprotein or polypeptide of the invention may have prophylactic use inclosed as well as open fracture reduction and also in the improvedfixation of artificial joints. De novo bone formation induced by anosteogenic agent contributes to the repair of congenital, traumainduced, or oncologic resection induced craniofacial defects, and alsois useful in cosmetic plastic surgery.

[0221] A protein or polypeptide of this invention may also be used inthe treatment and/or detection (e.g., a diagnostic) of periodontaldisease, and in other tooth repair processes. Such agents may provide anenvironment to attract bone-forming cells, stimulate growth ofbone-forming cells or induce differentiation of progenitors ofbone-forming cells. A protein or polypeptide of the invention may alsobe useful in the treatment of osteoporosis or osteoarthritis, such asthrough stimulation of bone and/or cartilage repair or by blockinginflammation or processes of tissue destruction (collagenase activity,osteoclast activity, etc.) mediated by inflammatory processes.

[0222] Another category of tissue regeneration activity that may beattributable to the protein or polypeptide of the present invention istendon/ligament formation. A protein or polypeptide of the presentinvention, which induces tendon/ligament-like tissue or other tissueformation in circumstances where such tissue is not normally formed, hasapplication in the healing of tendon or ligament tears, deformities andother tendon or ligament defects in humans and other animals. Such apreparation employing a tendon/ligament-like tissue inducing protein (asan antagonist or agonist) may have prophylactic use in preventing damageto tendon or ligament tissue, as well as use in the improved fixation oftendon or ligament to bone or other tissues, and in repairing defects totendon or ligament tissue. De novo tendon/ligament-like tissue formationinduced by a composition of the present invention contributes to therepair of congenital, trauma induced, or other tendon or ligamentdefects of other origin, and is also useful in cosmetic plastic surgeryfor attachment or repair of tendons or ligaments. The compositions ofthe present invention may provide environment to attract tendon- orligament-forming cells, stimulate growth of tendon- or ligament-formingcells, induce differentiation of progenitors of tendon- orligament-forming cells, or induce growth of tendon/ligament cells orprogenitors ex vivo for return in vivo to effect tissue repair. Thecompositions of the invention may also be useful in the treatment oftendinitis, carpal tunnel syndrome and other tendon or ligament defects.The compositions may also include an appropriate matrix and/orsequestering agent as a carrier as is well known in the art.

[0223] The protein or polypeptide of the present invention may also beuseful for proliferation of neural cells and for regeneration of nerveand brain tissue, i.e., for the treatment and/or detection (e.g., adiagnostic) of central and peripheral nervous system diseases andneuropathies, as well as mechanical and traumatic disorders, whichinvolve degeneration, death or trauma to neural cells or nerve tissue.More specifically, a protein or polypeptide may be used in the treatmentand/or detection (e.g., a diagnostic) of diseases of the peripheralnervous system, such as peripheral nerve injuries, peripheral neuropathyand localized neuropathies, and central nervous system diseases, such asAlzheimer's, Parkinson's disease, Huntington's disease, amyotrophiclateral sclerosis, and Shy-Drager syndrome. Further conditions which maybe treated in accordance with the present invention include mechanicaland traumatic disorders, such as spinal cord disorders, head trauma andcerebrovascular diseases such as stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using a protein or polypeptide of the invention.

[0224] Proteins or polypeptides of the invention may also be useful topromote better or faster closure of non-healing wounds, includingwithout limitation pressure ulcers, ulcers associated with vascularinsufficiency, surgical and traumatic wounds, and the like.

[0225] It is expected that a protein or polypeptide of the presentinvention may also exhibit activity for generation or regeneration ofother tissues, such as organs (including, for example, pancreas, liver,intestine, kidney, skin, endothelium), muscle (smooth, skeletal orcardiac) and vascular (including vascular endothelium) tissue, or forpromoting the growth of cells comprising such tissues. Part of thedesired effects may be by inhibition or modulation of fibrotic scarringto allow normal tissue to regenerate. A protein or polypeptide of theinvention may also exhibit angiogenic activity.

[0226] A protein or polypeptide of the present invention may also beuseful for gut protection or regeneration and treatment of lung or liverfibrosis, reperfusion injury in various tissues, and conditionsresulting from systemic cytokine damage.

[0227] A protein or polypeptide of the present invention may also beuseful for promoting or inhibiting differentiation of tissues describedabove from precursor tissues or cells; or for inhibiting the growth oftissues described above.

[0228] The activity of a protein or polypeptide of the invention may,among other means, be measured by the following methods:

[0229] Assays for tissue generation activity include, withoutlimitation, those described in: International Patent Publication No.WO95/16035 (bone, cartilage, tendon); International Patent PublicationNo. WO95/05846 (nerve, neuronal); International Patent Publication No.WO91/07491 (skin, endothelium).

[0230] Assays for wound healing activity include, without limitation,those described in: Winter, Epidermal Wound Healing, pps. 71-112(Maibach, H. I. and Rovee, D. T., eds.), Year Book Medical Publishers,Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol71:382-84 (1978).

[0231] A protein or polypeptide of the present invention may alsoexhibit agonist or antagonist activity against activin- orinhibin-related activities. Inhibins are characterized by their abilityto inhibit the release of follicle stimulating hormone (FSH), whileactivins and are characterized by their ability to stimulate the releaseof follicle stimulating hormone (FSH). Thus, a protein or polypeptide ofthe present invention that are agonists of inhibin, may be useful as acontraceptive based on the ability of inhibins to decrease fertility infemale mammals and decrease spermatogenesis in male mammals.Additionally, the proteins or polypeptides of the invention that areantagonists of activin, may be useful as a contraceptive based on theability of activin molecules in stimulating FSH release from cells ofthe anterior pituitary. Alternatively, the protein or polypeptide of theinvention that are agonists of activin, may be useful as a fertilityinducing therapeutic, based upon the ability of activin molecules instimulating FSH release from cells of the anterior pituitary. See, forexample, U.S. Pat. No. 4,798,885. Further, a proteins or polypeptides ofthe present invention that are antagonists of inhibin, may be useful asa fertility inducing therapeutic, based upon the ability of inhibins todecrease fertility in female mammals and decrease spermatogenesis inmale mammals. A protein or polypeptide of the invention may also beuseful for advancement of the onset of fertility in sexually immaturemammals, so as to increase the lifetime reproductive performance ofdomestic animals such as cows, sheep and pigs.

[0232] The activity of a protein or polypeptide of the invention may,among other means, be measured by the following methods:

[0233] Assays for activin/inhibin activity include, without limitation,those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling etal., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986;Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad.Sci. USA 83:3091-3095, 1986.

[0234] A protein or polypeptide of the present invention may be anantognist or agonist of chemotactic or chemokinetic activity (e.g., actas a chemokine) for mammalian cells, including, for example, monocytes,fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelialand/or endothelial cells. Chemotactic and chemokinetic proteins can beused to mobilize or attract a desired cell population to a desired siteof action. Antagonsits or agonists of chemotactic or chemokineticproteins provide particular advantages in treatment of inflammation, orwounds and other trauma to tissues, as well as in treatment of localizedinfections. For example, attraction of lymphocytes, monocytes orneutrophils to tumors or sites of infection may result in improvedimmune responses against the tumor or infecting agent.

[0235] A protein or polypeptide or peptide is an agonist of chemotacticactivity for a particular cell population if it can stimulate, directlyor indirectly, the directed orientation or movement of such cellpopulation. Preferably, the protein or polypeptide or peptide has theability to directly stimulate directed movement of cells. Whether aparticular protein has chemotactic activity for a population of cellscan be readily determined by employing such protein or peptide in anyknown assay for cell chemotaxis.

[0236] The activity of a protein or polypeptide of the invention may,among other means, be measured by the following methods:

[0237] Assays for chemotactic activity (which will identify proteinsthat induce or prevent chemotaxis) consist of assays that measure theability of a protein to induce the migration of cells across a membraneas well as the ability of a protein to induce the adhesion of one cellpopulation to another cell population. Suitable assays for movement andadhesion include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Marguiles, E. M. Shevach, W. Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 6.12, Measurement of alpha and betaChemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376,1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol.25:1744-1748; Gruber et al., J. of Immunol. 152:5860-5867, 1994;Johnston et al., J. of Immunol. 153:1762-1768, 1994.

[0238] A protein or polypeptide of the invention may also be anantagonist or agonist of hemostatic or thrombolytic activity. Such aprotein or polypeptide is expected to be useful in treatment and/ordetection (e.g., a diagnostic) of various coagulation disorders(including hereditary disorders, such as hemophilias) or to enhancecoagulation and other hemostatic events in treating wounds resultingfrom trauma, surgery or other causes. A protein or polypeptide of theinvention may also be useful for dissolving or inhibiting formation ofthromboses and for treatment and prevention of conditions resultingtherefrom (such as, for example, infarction of cardiac and centralnervous system vessels (e.g., stroke).

[0239] The activity of a protein or polypeptide of the invention may,among other means, be measured by the following methods:

[0240] Assay for hemostatic and thrombolytic activity include, withoutlimitation, those described in: Linet et al., J. Clin.Pharmacol.26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987;Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins35:467-474, 1988.

[0241] A protein or polypeptide of the present invention may alsodemonstrate activity as receptors, receptor ligands or antagonists oragonists of receptor/ligand interactions. Examples of such receptors andligands include, without limitation, cytokine receptors and theirligands, receptor kinases and their ligands, receptor phosphatases andtheir ligands, receptors involved in cell-cell interactions and theirligands (including without limitation, cellular adhesion molecules (suchas selecting, integrins and their ligands) and receptor/ligand pairsinvolved in antigen presentation, antigen recognition and development ofcellular and humoral immune responses). Receptors and ligands are alsouseful for screening of potential peptide or small molecule inhibitorsof the relevant receptor/ligand interaction. A protein or polypeptide ofthe present invention (including, without limitation, fragments ofreceptors and ligands) may themselves be useful as inhibitors ofreceptor/ligand interactions.

[0242] The activity of a protein or polypeptide of the invention may,among other means, be measured by the following methods:

[0243] Suitable assays for receptor-ligand activity include withoutlimitation those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28,Measurement of Cellular Adhesion under static conditions7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868,1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein etal., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol.Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.

[0244] Proteins or polypeptides of the present invention may also beantagonists or agonists of inflammation. Anti-inflammatory activity maybe achieved by providing a stimulus to cells involved in theinflammatory response, by inhibiting or promoting cell-cell interactions(such as, for example, cell adhesion), by inhibiting or promotingchemotaxis of cells involved in the inflammatory process, inhibiting orpromoting cell extravasation, or by stimulating or suppressingproduction of other factors which more directly inhibit or promote aninflammatory response. Proteins or polypeptides of the invention can beused to treat and/or detect (e.g., a diagnostic) inflammatory conditionsincluding chronic or acute conditions, including without limitationintimation associated with infection (such as septic shock, sepsis orsystemic inflammatory response syndrome (SIRS)), ischemia-reperfusioninjury, endotoxin lethality, arthritis, complement-mediated hyperacuterejection, nephritis, cytokine or chemokine-induced lung injury,inflammatory bowel disease, Crohn's disease or resulting from overproduction of cytokines such as TNF or IL-1. Proteins or polypeptides ofthe invention may also be useful to treat anaphylaxis andhypersensitivity to an antigenic substance or material.

[0245] Nervous system disorders, which can be treated and/or detected(e.g., a diagnostic) with the polypeptides or proteins of the inventioninclude but are not limited to nervous system injuries, and diseases ordisorders which result in either a disconnection of axons, a diminutionor degeneration of neurons, or demyelination. Nervous system lesionswhich may be treated and/or detected (e.g., a diagnostic) in a patient(including human and non-human mammalian patients) according to theinvention include but are not limited to the following lesions of eitherthe central (including spinal cord, brain) or peripheral nervoussystems:

[0246] (i) traumatic lesions, including lesions caused by physicalinjury or associated with surgery, for example, lesions which sever aportion of the nervous system, or compression injuries;

[0247] (ii) ischemic lesions, in which a lack of oxygen in a portion ofthe nervous system results in neuronal injury or death, includingcerebral infarction or ischemia, or spinal cord infarction or ischemia;

[0248] (iii) malignant lesions, in which a portion of the nervous systemis destroyed or injured by malignant tissue which is either a nervoussystem associated malignancy or a malignancy derived from non-nervoussystem tissue;

[0249] (iv) infectious lesions, in which a portion of the nervous systemis destroyed or injured as a result of infection, for example, by anabscess or associated with infection by human immunodeficiency virus,herpes zoster, or herpes simplex virus or with Lyme disease,tuberculosis, syphilis;

[0250] (v) degenerative lesions, in which a portion of the nervoussystem is destroyed or injured as a result of a degenerative processincluding but not limited to degeneration associated with Parkinson'sdisease, Alzheimer's disease, Huntington's chorea, or amyotrophiclateral sclerosis;

[0251] (vi) lesions associated with nutritional diseases or disorders,in which a portion of the nervous system is destroyed or injured by anutritional disorder or disorder of metabolism including but not limitedto, vitamin B12 deficiency, folic acid deficiency, Wernicke disease,tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primarydegeneration of the corpus callosum), and alcoholic cerebellardegeneration;

[0252] (vii) neurological lesions associated with systemic diseasesincluding but not limited to diabetes (diabetic neuropathy, Bell'spalsy), systemic lupus erythematosus, carcinoma, or sarcoidosis;

[0253] (viii) lesions caused by toxic substances including alcohol,lead, or particular neurotoxins; and

[0254] (ix) demyelinated lesions in which a portion of the nervoussystem is destroyed or injured by a demyelinating disease including butnot limited to multiple sclerosis, human immunodeficiencyvirus-associated myelopathy, transverse myelopathy or variousetiologies, progressive multifocal leukoencephalopathy, and centralpontine myelinolysis.

[0255] Therapeutics which are useful according to the invention fortreatment of a nervous system disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofneurons. For example, and not by way of limitation, therapeutics whichelicit any of the following effects may be useful according to theinvention:

[0256] (i) increased survival time of neurons in culture;

[0257] (ii) increased sprouting of neurons in culture or in vivo;

[0258] (iii) increased production of a neuron-associated molecule inculture or in vivo, e.g., choline acetyltransferase oracetylcholinesterase with respect to motor neurons; or

[0259] (iv) decreased symptoms of neuron dysfunction in vivo.

[0260] Such effects may be measured by any method known in the art. Inpreferred, non-limiting embodiments, increased survival of neurons maybe measured by the method set forth in Arakawa et al. (1990, J.Neurosci. 10:3507-3515); increased sprouting of neurons may be detectedby methods set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-82) orBrown et al. (1981, Ann. Rev. Neurosci. 4:17-42); increased productionof neuron-associated molecules may be measured by bioassay, enzymaticassay, antibody binding, Northern blot assay, etc., depending on themolecule to be measured; and motor neuron dysfunction may be measured byassessing the physical manifestation of motor neuron disorder, e.g.,weakness, motor neuron conduction velocity, or functional disability.

[0261] In a specific embodiments, motor neuron disorders that may betreated and/or detected (e.g., a diagnostic) according to the inventioninclude but are not limited to disorders such as infarction, infection,exposure to toxin, trauma, surgical damage, degenerative disease ormalignancy that may affect motor neurons as well as other components ofthe nervous system, as well as disorders that selectively affect neuronssuch as amyotrophic lateral sclerosis, and including but not limited toprogressive spinal muscular atrophy, progressive bulbar palsy, primarylateral sclerosis, infantile and juvenile muscular atrophy, progressivebulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis andthe post polio syndrome, and Hereditary Motorsensory Neuropathy(Charcot-Marie-Tooth Disease).

[0262] A protein or polypeptide of the invention may also exhibit one ormore of the following additional activities or effects: inhibiting thegrowth, infection or function of, or killing, infectious agents,including, without limitation, bacteria, viruses, fungi and otherparasites; effecting (suppressing or enhancing) bodily characteristicsor plant characteristics, including, without limitation, height, weight,hair color, eye color, skin, fat to lean ratio or other tissuepigmentation, or organ or body part size or shape (such as, for example,breast augmentation or diminution, change in bone form or shape);effecting biorhythms or caricadic cycles or rhythms; effecting thefertility of male or female subjects; effecting the metabolism,catabolism, anabolism, processing, utilization, storage or eliminationof dietary fat, lipid, protein, carbohydrate, vitamins, minerals,co-factors or other nutritional factors or component(s); effectingbehavioral characteristics, including, without limitation, appetite,libido, stress, cognition (including cognitive disorders), depression(including depressive disorders) and violent behaviors; providinganalgesic effects or other pain reducing effects; promotingdifferentiation and growth of embryonic stem cells in lineages otherthan hematopoietic lineages; hormonal or endocrine activity; in the caseof enzymes, correcting deficiencies of the enzyme and treatingdeficiency-related diseases; treatment of hyperproliferative disorders(such as, for example, psoriasis); immunoglobulin-like activity (suchas, for example, the ability to bind antigens or complement); and theability to act as an antigen in a vaccine composition to raise an immuneresponse against such protein or another material or entity which iscross-reactive with such protein.

[0263] A protein or polypeptide of the present invention (from whateversource derived, including without limitation from recombinant andnon-recombinant sources) may be administered to a patient in need, byitself, or in pharmaceutical compositions where it is mixed withsuitable carriers or excipient(s) at doses to treat or ameliorate avariety of disorders. Such a composition may also contain (in additionto protein or polypeptide and a carrier) diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials well known inthe art. The term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). The characteristics ofthe carrier will depend on the route of administration. Thepharmaceutical composition of the invention may also contain cytokines,lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF,thrombopoietin, stem cell factor, and erythropoietin. The pharmaceuticalcomposition may further contain other agents which either enhance theactivity of the protein or polypeptide or compliment its activity or usein treatment. Such additional factors and/or agents may be included inthe pharmaceutical composition to produce a synergistic effect withprotein or polypeptide of the invention, or to minimize side effects.Conversely, protein or polypeptide of the present invention may beincluded in formulations of the particular cytokine, lymphokine, otherhematopoietic factor, thrombolytic or anti-thrombotic factor, oranti-inflammatory agent to minimize side effects of the cytokine,lymphokine, other hematopoietic factor, thrombolytic or anti-thromboticfactor, or anti-inflammatory agent. A protein or polypeptide of thepresent invention may be active in multimers (e.g., heterodimers orhomodimers) or complexes with itself or other proteins. As a result,pharmaceutical compositions of the invention may comprise a protein orpolypeptide of the invention in such multimeric or complexed form.

[0264] Techniques for formulation and administration of the compounds ofthe instant application may be found in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition. Atherapeutically effective dose further refers to that amount of thecompound sufficient to result in amelioration of symptoms, e.g.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, a therapeutically effective dose refersto that ingredient alone. When applied to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, serially or simultaneously.

[0265] In practicing the method of treatment or use of the presentinvention, a therapeutically effective amount of protein or polypeptideof the present invention is administered to a mammal having a conditionto be treated. Protein or polypeptide of the present invention may beadministered in accordance with the method of the invention either aloneor in combination with other therapies such as treatments employingcytokines, lymphokines or other hematopoietic factors. Whenco-administered with one or more cytokines, lymphokines or otherhematopoietic factors, protein or polypeptide of the prese effectiveamount of protein or polypeptide of the present invention isadministered to a mammal having a condition to be treated. Protein orpolypeptide of the present invention may be administered in accordancewith the method of the invention either alone or in combination withother therapies such as treatments employing cytokines, lymphokines orother hematopoietic factors. When co-administered with one or morecytokines, lymphokines or other hematopoietic factors, protein orpolypeptide of the preseoutes of administration may, for example,include oral, rectal, transmucosal, or intestinal administration;parenteral delivery, including intramuscular, subcutaneous,intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections. Administration of protein or polypeptide of thepresent invention used in the pharmaceutical composition or to practicethe method of the present invention can be carried out in a variety ofconventional ways, such as oral ingestion, inhalation, topicalapplication or cutaneous, subcutaneous, intraperitoneal, parenteral orintravenous injection. Intravenous administration to the patient ispreferred.

[0266] Alternately, one may administer the compound in a local ratherthan systemic manner, for example, via injection of the compounddirectly into a arthritic joints or in fibrotic tissue, often in a depotor sustained release formulation. In order to prevent the scarringprocess frequently occurring as complication of glaucoma surgery, thecompounds may be administered topically, for example, as eye drops.Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with a specific antibody,targeting, for example, arthritic or fibrotic tissue. The liposomes willbe targeted to and taken up selectively by the afflicted tissue.

[0267] Pharmaceutical compositions for use in accordance with thepresent invention thus may be formulated in a conventional manner usingone or more physiologically acceptable carriers comprising excipientsand auxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. These pharmaceuticalcompositions may be manufactured in a manner that is itself known, e.g.,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Proper formulation is dependent upon the route ofadministration chosen. When a therapeutically effective amount ofprotein or polypeptide of the present invention is administered orally,protein or polypeptide of the present invention will be in the form of atablet, capsule, powder, solution or elixir. When administered in tabletform, the pharmaceutical composition of the invention may additionallycontain a solid carrier such as a gelatin or an adjuvant. The tablet,capsule, and powder contain from about 5 to 95% protein or polypeptideof the present invention, and preferably from about 25 to 90% protein orpolypeptide of the present invention. When administered in liquid form,a liquid carrier such as water, petroleum, oils of animal or plantorigin such as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils may be added. The liquid form of the pharmaceuticalcomposition may further contain physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol. When administered in liquidform, the pharmaceutical composition contains from about 0.5 to 90% byweight of protein or polypeptide of the present invention, andpreferably from about 1 to 50% protein or polypeptide of the presentinvention.

[0268] When a therapeutically effective amount of protein or polypeptideof the present invention is administered by intravenous, cutaneous orsubcutaneous injection, protein or polypeptide of the present inventionwill be in the form of a pyrogen-free, parenterally acceptable aqueoussolution. The preparation of such parenterally acceptable proteinsolutions, having due regard to pH, isotonicity, stability, and thelike, is within the skill in the art. A preferred pharmaceuticalcomposition for intravenous, cutaneous, or subcutaneous injection shouldcontain, in addition to protein or polypeptide of the present invention,an isotonic vehicle such as Sodium Chloride Injection, Ringer'sInjection, Dextrose Injection, Dextrose and Sodium Chloride Injection,Lactated Ringer's Injection, or other vehicle as known in the art. Thepharmaceutical composition of the present invention may also containstabilizers, preservatives, buffers, antioxidants, or other additivesknown to those of skill in the art. For injection, the agents of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

[0269] For oral administration, the compounds can be formulated readilyby combining the active compounds with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

[0270] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration. For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner.

[0271] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. The compounds maybe formulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

[0272] Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0273] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. In additionto the formulations described previously, the compounds may also beformulated as a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

[0274] A pharmaceutical carrier for the hydrophobic compounds of theinvention is a cosolvent system comprising benzyl alcohol, a nonpolarsurfactant, a water-miscible organic polymer, and an aqueous phase. Thecosolvent system may be the VPD co-solvent system. VPD is a solution of3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80,and 65% w/v polyethylene glycol 300, made up to volume in absoluteethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1with a 5% dextrose in water solution. This co-solvent system dissolveshydrophobic compounds well, and itself produces low toxicity uponsystemic administration. Naturally, the proportions of a co-solventsystem may be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the co-solventcomponents may be varied: for example, other low-toxicity nonpolarsurfactants may be used instead of polysorbate 80; the fraction size ofpolyethylene glycol may be varied; other biocompatible polymers mayreplace polyethylene glycol, e.g., polyvinyl pyrrolidone; and othersugars or polysaccharides may substitute for dextrose. Alternatively,other delivery systems for hydrophobic pharmaceutical compounds may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Certain organic solventssuch as dimethylsulfoxide also may be employed, although usually at thecost of greater toxicity. Additionally, the compounds may be deliveredusing a sustained-release system, such as semi-permeable matrices ofsolid hydrophobic polymers containing the therapeutic agent. Various ofsustained-release materials have been established and are well known bythose skilled in the art. Sustained-release capsules may, depending ontheir chemical nature, release the compounds for a few weeks up to over100 days. Depending on the chemical nature and the biological stabilityof the therapeutic reagent, additional strategies for proteinstabilization may be employed.

[0275] The pharmaceutical compositions also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols. Many of the proteinase inhibitingcompounds of the invention may be provided as salts withpharmaceutically compatible counterions. Such pharmaceuticallyacceptable base addition salts are those salts which retain thebiological effectiveness and properties of the free acids and which areobtained by reaction with inorganic or organic bases such as sodiumhydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine,monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate,triethanol amine and the like.

[0276] The pharmaceutical composition of the invention may be in theform of a complex of the protein(s) or polypeptide(s)of presentinvention along with protein or peptide antigens. The protein and/orpeptide antigen will deliver a stimulatory signal to both B and Tlymphocytes. B lymphocytes will respond to antigen through their surfaceimmunoglobulin receptor. T lymphocytes will respond to antigen throughthe T cell receptor (TCR) following presentation of the antigen by MHCproteins. MHC and structurally related proteins including those encodedby class I and class II MHC genes on host cells will serve to presentthe peptide antigen(s) to T lymphocytes. The antigen components couldalso be supplied as purified MHC-peptide complexes alone or withco-stimulatory molecules that can directly signal T cells. Alternativelyantibodies able to bind surface immunoglobulin and other molecules on Bcells as well as antibodies able to bind the TCR and other molecules onT cells can be combined with the pharmaceutical composition of theinvention. The pharmaceutical composition of the invention may be in theform of a liposome in which protein or polypeptide of the presentinvention is combined, in addition to other pharmaceutically acceptablecarriers, with amphipathic agents such as lipids which exist inaggregated form as micelles, insoluble monolayers, liquid crystals, orlamellar layers in aqueous solution. Suitable lipids for liposomalformulation include, without limitation, monoglycerides, diglycerides,sulfatides, lysolecithin, phospholipids, saponin, bile acids, and thelike. Preparation of such liposomal formulations is within the level ofskill in the art, as disclosed, for example, in U.S. Pat. Nos.4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which areincorporated herein by reference.

[0277] The amount of protein or polypeptide of the present invention inthe pharmaceutical composition of the present invention will depend uponthe nature and severity of the condition being treated, and on thenature of prior treatments which the patient has undergone. Ultimately,the attending physician will decide the amount of protein or polypeptideof the present invention with which to treat each individual patient.Initially, the attending physician will administer low doses of proteinor polypeptide of the present invention and observe the patient'sresponse. Larger doses of protein or polypeptide of the presentinvention may be administered until the optimal therapeutic effect isobtained for the patient, and at that point the dosage is not increasedfurther. It is contemplated that the various pharmaceutical compositionsused to practice the method of the present invention should containabout 0.01 μg to about 100 mg (preferably about 0.1 μg to about 10 mg,more preferably about 0.1 μg to about 1 mg) of protein or polypeptide ofthe present invention per kg body weight. For compositions of thepresent invention which are useful for bone, cartilage, tendon orligament regeneration, the therapeutic method includes administering thecomposition topically, systematically, or locally as an implant ordevice. When administered, the therapeutic composition for use in thisinvention is, of course, in a pyrogen-free, physiologically acceptableform. Further, the composition may desirably be encapsulated or injectedin a viscous form for delivery to the site of bone, cartilage or tissuedamage. Topical administration may be suitable for wound healing andtissue repair. Therapeutically useful agents other than a protein orpolypeptide of the invention which may also optionally be included inthe composition as described above, may alternatively or additionally,be administered simultaneously or sequentially with the composition inthe methods of the invention. Preferably for bone and/or cartilageformation, the composition would include a matrix capable of deliveringthe protein-containing composition to the site of bone and/or cartilagedamage, providing a structure for the developing bone and cartilage andoptimally capable of being resorbed into the body. Such matrices may beformed of materials presently in use for other implanted medicalapplications.

[0278] The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalciumphosphate. The bioceramics may be altered in composition, suchas in calcium-aluminate-phosphate and processing to alter pore size,particle size, particle shape, and biodegradability. Presently preferredis a 50:50 (mole weight) copolymer of lactic acid and glycolic acid inthe form of porous particles having diameters ranging from 150 to 800microns. In some applications, it will be useful to utilize asequestering agent, such as carboxymethyl cellulose or autologous bloodclot, to prevent the protein or polypeptide compositions fromdisassociating from the matrix.

[0279] A preferred family of sequestering agents is cellulosic materialssuch as alkylcelluloses (including hydroxyalkylcelluloses), includingmethylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropyl-methylcellulose, andcarboxymethylcellulose, the most preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). Theamount of sequestering agent useful herein is 0.5-20 wt %, preferably1-10 wt % based on total formulation weight, which represents the amountnecessary to prevent desorbtion of the protein or polypeptide from thepolymer matrix and to provide appropriate handling of the composition,yet not so much that the progenitor cells are prevented frominfiltrating the matrix, thereby providing the protein or polypeptidethe opportunity to assist the osteogenic activity of the progenitorcells. In further compositions, proteins or polypeptides of theinvention may be combined with other agents beneficial to the treatmentof the bone and/or cartilage defect, wound, or tissue in question. Theseagentsminclude various growth factors such as epidermal growth factor(EGF), platelet derived growth factor (PDGF), transforming growthfactors (TGF-.alphA. and TGF-.beta.), and insulin-like growth factor(IGF).

[0280] The therapeutic compositions are also presently valuable forveterinary applications. Particularly domestic animals and thoroughbredhorses, in addition to humans, are desired patients for such treatmentwith proteins or polypeptides of the present invention. The dosageregimen of a protein-containing pharmaceutical composition to be used intissue regeneration will be determined by the attending physicianconsidering various factors which modify the action of the proteins orpolypeptides, e.g., amount of tissue weight desired to be formed, thesite of damage, the condition of the damaged tissue, the size of awound, type of damaged tissue (e.g., bone), the patient's age, sex, anddiet, the severity of any infection, time of administration and otherclinical factors. The dosage may vary with the type of matrix used inthe reconstitution and with inclusion of other proteins in thepharmaceutical composition. For example, the addition of other knowngrowth factors, such as IGF I (insulin like growth factor I), to thefinal composition, may also effect the dosage. Progress can be monitoredby periodic assessment of tissue/bone growth and/or repair, for example,X-rays, histomorphometric determinations and tetracycline labeling.

[0281] Polynucleotides of the present invention can also be used forgene therapy. Such polynucleotides can be introduced either in vivo orex vivo into cells for expression in a mammalian subject.Polynucleotides of the invention may also be administered by other knownmethods for introduction of nucleic acid into a cell or organism(including, without limitation, in the form of viral vectors or nakedDNA).

[0282] Cells may also be cultured ex vivo in the presence of proteins orpolypeptides of the present invention in order to proliferate or toproduce a desired effect on or activity in such cells. Treated cells canthen be introduced in vivo for therapeutic purposes.

[0283] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve its intended purpose. Morespecifically, a therapeutically effective amount means an amounteffective to prevent development of or to alleviate the existingsymptoms of the subject being treated. Determination of the effectiveamounts is well within the capability of those skilled in the art,especially in light of the detailed disclosure provided herein. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Forexample, a dose can be formulated in animal models to achieve acirculating concentration range that includes the IC₅₀ as determined incell culture (i.e., the concentration of the test compound whichachieves a half-maximal inhibition of the C-proteinase activity). Suchinformation can be used to more accurately determine useful doses inhumans.

[0284] A therapeutically effective dose refers to that amount of thecompound that results in amelioration of symptoms or a prolongation ofsurvival in a patient. Toxicity and therapeutic efficacy of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio between LD₅₀ and ED₅₀. Compounds whichexhibit high therapeutic indices are preferred. The data obtained fromthese cell culture assays and animal studies can be used in formulatinga range of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. See, e.g., Fingl et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p.1.Dosage amount and interval may be adjustedindividually to provide plasma levels of the active moiety which aresufficient to maintain the C-proteinase inhibiting effects, or minimaleffective concentration (MEC). The MEC will vary for each compound butcan be estimated from in vitro data; for example, the concentrationnecessary to achieve 50-90% inhibition of the C-proteinase using theassays described herein. Dosages necessary to achieve the MEC willdepend on individual characteristics and route of administration.However, HPLC assays or bioassays can be used to determine plasmaconcentrations.

[0285] Dosage intervals can also be determined using MEC value.Compounds should be administered using a regimen which maintains plasmalevels above the MEC for 10-90% of the time, preferably between 30-90%and most preferably between 50-90%. In cases of local administration orselective uptake, the effective local concentration of the drug may notbe related to plasma concentration.

[0286] The amount of composition administered will, of course, bedependent on the subject being treated, on the subject's weight, theseverity of the affliction, the manner of administration and thejudgment of the prescribing physician.

[0287] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.Compositions comprising a compound of the invention formulated in acompatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labelled for treatment of an indicatedcondition.

[0288] In one application of this embodiment, a nucleotide sequence ofthe present invention can be recorded on computer readable media. Asused herein, ‘computer readable media’ refers to any medium which can beread and accessed directly by a computer. Such media include, but arenot limited to: magnetic storage media, such as floppy discs, hard discstorage medium, and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. A skilled artisan canreadily appreciate how any of the presently known computer readablemediums can be used to create a manufacture comprising computer readablemedium having recorded thereon a nucleotide sequence of the presentinvention. As used herein, ‘recorded’ refers to a process for storinginformation on computer readable medium. A skilled artisan can readilyadopt any of the presently known methods for recording information oncomputer readable medium to generate manufactures comprising thenucleotide sequence information of the present invention.

[0289] A variety of data storage structures are available to a skilledartisan for creating a computer readable medium having recorded thereona nucleotide sequence of the present invention. The choice of the datastorage structure will generally be based on the means chosen to accessthe stored information. In addition, a variety of data processorprograms and formats can be used to store the nucleotide sequenceinformation of the present invention on computer readable medium. Thesequence information can be represented in a word processing text file,formatted in commercially-available software such as WordPerfect andMicrosoft Word, or represented in the form of an ASCII file, stored in adatabase application, such as DB2, Sybase, Oracle, or the like. Askilled artisan can readily adapt any number of dataprocessorstructuring formats (e.g. text file or database) in order to obtaincomputer readable medium having recorded thereon the nucleotide sequenceinformation of the present invention. Computer software is publiclyavailable which allows a skilled artisan to access sequence informationprovided in a computer readable medium. The examples which followdemonstrate how software which implements the BLAST (Altschul et al., J.Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem.17:203-207 (1993)) search algorithms on a Sybase system is used toidentify open reading frames (ORFs) within a nucleic acid sequence. SuchORFs may be protein or polypeptide encoding fragments and may be usefulin producing commercially important protein or polypeptides such asenzymes used in fermentation reactions and in the production ofcommercially useful metabolites.

[0290] As used herein, ‘a computer-based system’ refers to the hardwaremeans, software means, and data storage means used to analyze thenucleotide sequence information of the present invention. The minimumhardware means of the computer-based systems of the present inventioncomprises a central processing unit (CPU), input means, output means,and data storage means. A skilled artisan can readily appreciate thatany one of the currently available computer-based systems are suitablefor use in the present invention. As stated above, the computer-basedsystems of the present invention comprise a data storage means havingstored therein a nucleotide sequence of the present invention and thenecessary hardware means and software means for supporting andimplementing a search means. As used herein, ‘data storage means’ refersto memory which can store nucleotide sequence information of the presentinvention, or a memory access means which can access manufactures havingrecorded thereon the nucleotide sequence information of the presentinvention.

[0291] As used herein, ‘search means’ refers to one or more programswhich are implemented on the computer-based system to compare a targetsequence or target structural motif with the sequence information storedwithin the data storage means. Search means are used to identifyfragments or regions of a known sequence which match a particular targetsequence or target motif. A variety of known algorithms are disclosedpublicly and a variety of commercially available software for conductingsearch means are and can be used in the computer-based systems of thepresent invention. Examples of such software includes, but is notlimited to, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). Askilled artisan can readily recognize that any one of the availablealgorithms or implementing software packages for conducting homologysearches can be adapted for use in the present computer-based systems.As used herein, a ‘target sequence’ can be any nucleic acid or aminoacid sequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. The most preferred sequence length of atarget sequence is from about 10 to 100 amino acids or from about 30 to300 nucleotide residues. However, it is well recognized that searchesfor commercially important fragments, such as sequence fragmentsinvolved in gene expression and protein or polypeptide processing, maybe of shorter length.

[0292] As used herein, ‘a target structural motif,’ or ‘target motif,’refers to any rationally selected sequence or combination of sequencesin which the sequence(s) are chosen based on a three-dimensionalconfiguration which is formed upon the folding of the target motif.There are a variety of target motifs known in the art. Protein orpolypeptide target motifs include, but are not limited to, enzyme activesites and signal sequences. Nucleic acid target motifs include, but arenot limited to, promoter sequences, hairpin structures and inducibleexpression elements (protein or polypeptide binding sequences).

[0293] The present invention further provides methods to identify thepresence or expression of one of the targets recognized by a polypeptideor protein of the present invention, or homolog thereof, in a testsample.

[0294] In general, methods for detecting a target recognized by apolypeptide or protein of the invention can comprise contacting a samplewith a polypeptide or protein of the invention that binds to and forms acomplex with the target for a period sufficient to form a complex, anddetecting the complex, so that if a complex is detected, a target of theinvention is detected in the sample.

[0295] In detail, such methods comprise incubating a test sample withone or more of the antibodies of the present invention and assaying forbinding of the antibodies to the target within the test sample.

[0296] Conditions for incubating an antibody, including a Fab fragmentof the invention, with a test sample vary. Incubation conditions dependon the format employed in the assay, the detection methods employed, andthe type and nature of the antibody used in the assay. One skilled inthe art will recognize that any one of the commonly availableamplification or immunological assay formats can readily be adapted toemploy the antibodies of the present invention. Examples of such assayscan be found in Chard, T., An Introduction to Radioimmunoassay andRelated Techniques, Elsevier Science Publishers, Amsterdam, TheNetherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985); and Kuwata etal., BioChem. Biophys. Res. Commun. 245:764-73 (1998), Hillenkamp etal., Anal. Chem. 63:1193-202 (1991), U.S. Pat. Nos. 5,111,937 and5,719,060. The test samples of the present invention include cells,protein or polypeptide or membrane extracts of cells, or biologicalfluids such as sputum, blood, serum, plasma, or urine. The test sampleused in the above-described method will vary based on the assay format,nature of the detection method and the tissues, cells or extracts usedas the sample to be assayed. Methods for preparing protein orpolypeptide extracts or membrane extracts of cells are well known in theart and can be readily be adapted in order to obtain a sample which iscompatible with the system utilized.

[0297] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention. Specifically, the invention provides a compartmentkit to receive, in close confinement, one or more containers whichcomprises: (a) a first container comprising one of the antibodies of thepresent invention; and (b) one or more other containers comprising oneor more of the following: wash reagents, reagents capable of detectingpresence of a bound antibody.

[0298] In detail, a compartment kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers or strips of plastic or paper. Suchcontainers allows one to efficiently transfer reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother. Such containers will include a container which will accept thetest sample, a container which contains the antibodies used in theassay, containers which contain wash reagents (such as phosphatebuffered saline, Tris-buffers, etc.), and containers which contain thereagents used to detect the bound antibody or probe. Types of detectionreagents include labeled secondary antibodies, or in the alternative, ifthe primary antibody is labeled, the enzymatic, or antibody bindingreagents which are capable of reacting with the labeled antibody. Oneskilled in the art will readily recognize that the disclosed probes andantibodies of the present invention can be readily incorporated into oneof the established kit formats which are well known in the art.

[0299] Using the polypeptides or proteins of the invention, the presentinvention further provides methods of obtaining and identifying agentswhich bind to a target recognized by the polypeptide or protein. Indetail, said method comprises the steps of:

[0300] (a) contacting a target with an isolated protein or polypeptideof the present invention; and

[0301] (b) determining whether the target binds to said protein orpolypeptide.

[0302] In general, such methods for identifying compounds that bind to apolypeptide of the invention can comprise contacting a compound with apolypeptide of the invention for a time sufficient to form apolypeptide/compound complex, and detecting the complex, so that if apolypeptide/compound complex is detected, a compound that binds to apolynucleotide of the invention is identified.

[0303] Methods for identifying compounds that bind to a polypeptide ofthe invention can also comprise contacting a compound with a polypeptideof the invention in a cell for a time sufficient to form apolypeptide/compound complex, wherein the complex drives expression of areceptor gene sequence in the cell, and detecting the complex bydetecting reporter gene sequence expression, so that if apolypeptide/compound complex is detected, a compound that binds apolypeptide of the invention is identified.

[0304] Compounds identified via such methods can include compounds whichmodulate the activity of a target recognized by a polypeptide or proteinof the invention (that is, increase or decrease the target's activity,relative to activity observed in the absence of the compound).Alternatively, compounds identified via such methods can includecompounds which modulate the expression of a polynucleotide of theinvention (that is, increase or decrease expression relative toexpression levels observed in the absence of the compound). Compounds,such as compounds identified via the methods of the invention, can betested using standard assays well known to those of skill in the art fortheir ability to modulate activity/expression.

[0305] The agents screened in the above assay can be, but are notlimited to, peptides, carbohydrates, vitamin derivatives, or otherpharmaceutical agents. The agents can be selected and screened at randomor rationally selected or designed using protein modeling techniques.

[0306] For random screening, agents such as peptides, carbohydrates,pharmaceutical agents and the like are selected at random and areassayed for their ability to bind to the target recognized by thepolypeptide or protein of the present invention. Alternatively, agentsmay be rationally selected or designed. As used herein, an agent is saidto be ‘rationally selected or designed’ when the agent is chosen basedon the configuration of the particular protein or polypeptide. Forexample, one skilled in the art can readily adapt currently availableprocedures to generate peptides, pharmaceutical agents and the likecapable of binding to a specific peptide sequence in order to generaterationally designed antipeptide peptides, for example see Hurby et al.,Application of Synthetic Peptides: Antisense Peptides,’ In SyntheticPeptides, A User's Guide, W.H. Freeman, N.Y. (1992), pp. 289-307, andKaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceuticalagents, or the like.

[0307] In addition to the foregoing, one class of agents of the presentinvention, as broadly described, can be used to control gene expressionthrough binding to one of the ORFs or EMFs of the present invention. Asdescribed above, such agents can be randomly screened or rationallydesigned/selected. Targeting the ORF or EMF allows a skilled artisan todesign sequence specific or element specific agents, modulating theexpression of either a single ORF or multiple ORFs which rely on thesame EMF for expression control.

[0308] As choice of antibody format, we preferred the Fab format abovethe scFv format, because the Fab format allows rapid high through-putaffinity-screening assays for crude antibody preparations. Many scFv'sindeed form higher molecular weight species including dimers (Weidner,et al., (1992) J. Biol. Chem. 267, 10281-10288; Holliger, et al., (1993)Proc. Natl. Acad. Sci. U.S.A. 90, 6444-6448) and trimers (Korttet al.,(1997) Protein Eng. 10, 423-433), which complicate both selection andcharacterisation. We chose the Fab display format in which a variabledomain from a heavy or light chain gene is linked to a phage coatprotein, and in some embodiments, also carries a tag for detection andpurification. The other chain is expressed as separate fragment secretedinto the periplasm, where it can pair with the gene that is in a proteinfusion with the phage coat protein (Hoogenboom, et al., (1991) NucleicAcids Res. 19, 4133-4137). In some embodiments, the phage coat protein apIII coat protein. In other embodiments, the variable domain from aheavy chain gene is fused to the phage coat protein and the light chaingene is expressed as a separate fragment.

[0309] The choice for the Fab format was based on the notion that themonomeric appearance of the Fab permits the rapid screening of largenumbers of clones for kinetics of binding (off-rate) with crude proteinfractions. This reduces the time for post-selection analysisdramatically when compared to that needed for selected single-chain Fv(scFv) antibodies from phagemid libraries (Vaughan, et al., (1996) Nat.Biotechnol. 14, 309-314; Sheets, et al., (1998) Proc. Natl. Acad. Sci.U.S.A. 95, 6157-6162), or Fab fragments from other phage libraries(Griffiths, et al., (1993) EMBO J. 12, 725-734).

[0310] The Fab library of the invention produced on average 14 differentFab's against 6 antigens that were tested. These include tetanus toxoid,the hapten phenyl-oxazolone, the breast cancer associated MUC1 antigenand three highly related glycoprotein hormones: human ChorionicGonadotropin (‘hCG’), human Luteinizing Hormone (‘hLH’) and humanFollicle Stimulating Hormone (‘hFSH’). For the glycoprotein hormones,the Fab library of the invention produced a panel of eitherhomone-specific or cross-reactive antibodies. Thus, without usingsophisticated selection protocols, hormone specific as well ascross-reactive Fab's were retrieved against these highly homologousglycohormones, demonstrating that the library is a rich source ofantibody specificities. The affinities of the anti-glycohormoneantibodies varied between 2.7 and 38 nM. Finally, the Fab-format indeedpermitted the rapid screening and a reliable ranking of individualclones based on off-rate using crude fractions.

[0311] Furthermore, the specificities of the antibodies obtained byselections on the gonadotropins are unique: due to the high degree ofhomology between hLH and hCG it has been very difficult to isolate hCGspecific monoclonal antibodies with the hybridoma technology, whereasthere are very few hLH specific antibodies (Moyle, et al., (1990) J.Biol. Chem. 265, 8511-8518; Cole, (1997) Clin. Chem. 43, 2233-2243).Using a straight forward selection procedure, taking no precaution toavoid the selection of cross-reactive Fab's, we have readily isolatedfragments with all possible specificities: Fab's specific for any of thethree hormones hCG, hLH and hFSH, and cross-reactive Fab's recognizingthe common α-chain or epitopes on the β-chain shared by hCG and hLH.These selections demonstrated that antibodies directed against differentepitopes within single antigen molecules can be retrieved from thelibrary. The Fab library of the invention permits the monitoring ofselections with polyclonal phage preparations and large scale screeningof antibody off-rates with unpurified Fab fragments.

[0312] Overall, antibodies with off-rates in the order of 10⁻² to 10⁻⁴s⁻¹ and affinities up to 2.7 nM, were recovered. The kinetics of thesephage antibodies are of the same order of magnitude as antibodiesassociated with a secondary immune response.

[0313] An indication that antibodies from the Fab library behavesimilarly or better than antibodies from a scFv library with regards toaffinity comes from a comparison of selections of two differentlibraries on the same two antigens under identical conditions.Antibodies to MUC1 selected from a large naVve scFv library (Henderikxet al., (1998) Cancer Res. 58, 4324-4332) have faster off-rates then theequivalent Fab's isolated from the library described in this study.Further, they show a very distinct V-gene usage and have a differentfine specificity. Similarly, when comparing the off-rates of phageantibodies against the pancarcinoma marker Epithelial Glycoprotein-2,one of the Fab's selected from the present library appears to have a10-fold slower off-rate than the best scFv (Vaughan et al., (1996) Nat.Biotechnol. 14, 309-314).

[0314] The affinities of the selected antibody fragments is, however,very much dependent on the antigen used for selection. Sheets andcolleagues reported an affinity varying between 26 and 71 nM for theselected scFv fragments specific for the anti-Clostridia botulinumneurotoxin type A fragments, whereas for antibodies to the extracellulardomain of human ErbB-2, K_(d)'s between 0.22 and 4.03 nM were found(Sheets et al., (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6157-6162). Theaffinities of the gonadotropin specific Fab's selected from our libraryvaried between 2.7 and 38 nM, which is comparable to the protein bindingscFv's from the naVve library made by Vaughan et al. and Sheets et al.,and approaches the values of the best antibodies in their kind.

[0315] The size of the Fab library of the invention is not onlyimportant for affinity, but it also determines the success rate ofselection of antibodies against a large set of different antigens. Inthis respect the Fab library of the invention performs very well: over24 antibodies to the hapten phOx, and on average 13 antibodies againstthe other antigens were selected.

[0316] In the limited set of 14 Fab clones that were sequenced, weidentify antibodies with variable region genes from all large V-genefamilies, including V_(H1/3/4), V_(61/3), and V_(81/2), but also lessfrequently used segments of family V_(H6), V_(62/7) and V₈₇ wereretrieved. Most likely the use of an extended set of variable regiongene primers, designed on the most recent sequence information of thegermline V-regions, and/or the separate PCRs, combined with partiallyseparate cloning, ensured access to a highly diverse sample of the humanV-gene repertoire.

[0317] According to the present invention, a library is prepared frompolynucleotides which are capable of encoding the desired specificbinding pair member. A variety of techniques exist for preparing thelibrary, which may be prepared, for example, from either genomic DNA orcDNA. See, e.g., Sambrook et al., Molecular Cloning, A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989, which is incorporated herein by reference. Cells mayserve as the source of the polynucleotides which encode the specificbinding pair members of interest. Enrichment procedures and means foramplifying the regions containing the gene(s), may be employed. Forinstance, when the desired specific binding pair member is an antibody,RNA and or genomic DNA may be prepared, for example, from spleen cellsobtained from an unimmunized animal, from an animal immunized withtarget(s) of interest, from hybridoma cells, or from lymphoblastoidcells. The library of antibodies obtained from the unimmunized animalscontain an unbiased representation of the entire antibody repertoire,while the library of antibodies obtained from the immunized animalscontain a biased population of antibodies directed against epitopes ofthe target(s). Spleen cells, or immune cells from other tissues or thecirculatory system may be obtained from a variety of animal species,such as human, mouse, rat, equine, bovine, avian, etc.

[0318] Amplification of messenger RNA (mRNA) isolated from cells ofinterest, such as spleen or hybridoma cells, may be performed accordingto protocols outlined in, e.g., U.S. Pat. No. 4,683,202, Orlandi, et al.Proc. Natl. Acad. Sci. USA 86:3833-3837 (1989), Sastry et al., Proc.Natl. Acad. Sci. USA 86:5728-5732 (1989), and Huse et al. Science246:1275-1281 (1989), Abelson, J. and Simon, M. (eds), Methods inEnzymology, combinatorial chemistry, Vol. 267, San Diego: Academic Press(1996), Kay, B. K., Winter, J., McCafferty, J. (eds), Phage Display ofpeptides and Proteins, a Laboratory Manual, San Diego: Academic Press(1996), each incorporated herein by reference. oligonucleotide primersuseful in amplification protocols may be unique or degenerate orincorporate inosine at degenerate positions. Thus, for multi-chainimmunoglobulins, primers would be generally used for amplification ofsequences encoding the variable regions of both the heavy and lightchains. Restriction endonuclease recognition sequences may beincorporated into the primers to allow for the cloning of the amplifiedfragment into a vector in a predetermined reading frame for expression.

[0319] Expression libraries containing the amplified cDNA are typicallyprepared in a vector such as a bacteriophage or phagemid. Thecharacteristics of the suitable bacteriophage or phagemid depends on thespecific embodiment employed, and will generally be those whichconveniently allow insertion of the recombinant polynucleotides intohost cells by in vitro packaging or transformation.

[0320] Host cells are then infected with the phage or phagemid andhelper phage, and cultivated under conditions allowing for theexpression and assembly of phage particles. In one embodiment, theappropriate host cells for the bacteriophage or phagemids of theinvention are various strains of E. coli, specific examples depending onwhich of the several suitable vectors is chosen. Of course, phage orphagemid having bacterial hosts other than E. coli may also be used.

[0321] To enrich for and isolate phage particles or phage which containcloned library sequences that encode a desired specific binding pairmember, and thus to ultimately isolate the nucleic acid sequencesthemselves, phage particles or phage harvested from the host cells areaffinity purified. A target or binding partner for the desired specificbinding pair member is used in the affinity purification. For example,when the desired specific binding pair member is an antibody whichspecifically binds a particular target, the target is used to retrievephage particles or phage having the desired antibody on its outersurface. The target is typically adsorbed to an insoluble substrate,such as a particle or bead or plate. The phage particles or phage soobtained may then be amplified by infecting into host cells (with helperphage for the phage particles containing the phagemids). Additionalrounds of affinity enrichment and amplification may be employed untilthe desired level of enrichment is reached or the desired phageparticles or phage are no longer enriched relative to the backgroundphage particles or phage.

[0322] The enriched antibody-phage particles or phage are also screenedwith additional detection techniques such as expression plaque (orcolony) lift (see, e.g., Young and Davis, Science, 222:778-782 (1983),incorporated herein by reference) whereby the same or another bindingpartner is used as a probe. Screening may employ additional assays (fora catalytic activity, for example) which are used to detect, in situ,plaques expressing specific binding pair members having the desiredcharacteristics. The phage particles or phage obtained from thescreening protocol are infected into cells, propagated, and the phageparticle or phage DNA isolated and sequenced, and/or recloned into avector intended for gene expression in prokaryotes or eukaryotes toobtain larger amounts of the selected specific binding pair member.

[0323] In another embodiment, the specific binding pair member encodedin the library (or multiple chains comprising said specific binding pairmember) is transported to an extra-cytoplasmic compartment of the hostcell, usually the periplasmic space, to facilitate processing and/orproper assembly. When extra-cytoplasmic transport of the desiredspecific binding pair member is employed, the sequences encoding thespecific binding pair member are cloned adjacent to appropriatetranscriptional and translational signals and signal peptide leadersthat will direct the mature chains to the periplasm. As above, at leastone of the chains is cloned as a fusion protein with a phage coatprotein so that the phage coat protein does not substantially interferewith the ability of the specific binding pair member of interest to binda target which is used in the affinity enrichment protocol.

[0324] A preferred example of this embodiment is the placement of aspecific binding pair member in the N-terminus region of the minor coatprotein pIII of bacteriophage fd. Before incorporation into the phage,pIII resides in the inner membrane of the host cell with its N-terminusprotruding into the periplasm. In this configuration the polypeptide ofa specific binding pair member in the N-terminus of pIII is availablefor binding to other polypeptide chains that make up the specificbinding pair member of interest. This complex is then incorporated intothe mature phage particle or phage as it exits the cell and theC-terminus embeds in the coat of the phage particle or phage.

[0325] In this embodiment the synthesis and amplification ofpolynucleotides is as described above, and then is cloned into or near avector sequence encoding a coat protein, where the vector is, or isderived from, a filamentous phage, such as f1, fd, Pf1, M13, etc. In apreferred embodiment the filamentous phage is fd-tet. The phage vectoris chosen to contain a cloning site located in the 5′ region of a geneencoding a phage coat protein, such as, for example, the pIII coatprotein. An appropriate vector (e.g., fd-tet B1 which is describedbelow) allows oriented cloning of foreign sequences so that they areexpressed at or near the N-terminus of the mature coat protein.

[0326] A library is constructed by cloning the polynucleotides (e.g.,the V_(H) region) from the donor cells into a coat protein gene (e.g.,gene III, “gIII”) cloning site. The cloned sequences of, for example,the V_(H) domains are ultimately expressed as polypeptides or proteinsfused to the N-terminus of the mature coat protein on the outer,accessible surface of the assembled phage particles or phage.

[0327] When the desired protein is a multi-chain protein, such as anantibody or binding fragment thereof, the polynucleotide encoding thechain(s) not cloned into a phage coat protein may be cloned directlyinto an appropriate site (as described below) of the vector containingthe first chain-coat protein library; or, preferably, the subsequentchain(s) may be cloned as a separate library in a different plasmidvector, amplified, and subsequently the fragments installed in the firstchain-coat protein library vector. For example, when the first chain isan antibody heavy chain or binding fragment thereof, the ultimatedestination of light chain V_(L) cDNA sequence is in a vector thatalready contains a V_(H) sequence in a coat protein gene, thus randomlyrecombining V_(H) and V_(L) sequences in a single vector.

[0328] The second or subsequent chain of the desired multi-chainprotein, such as V_(L), is cloned so that it is expressed with a signalpeptide leader sequence that will direct its secretion into theperiplasm of the host cell. For example, several leader sequences havebeen shown to direct the secretion of antibody sequences in E. coli,such as OmpA (Hsiung, et al., Biotechnology 4:991-995 (1986)), pe1B(Better, et al., Science 240:1041-1043 (1988)), phoA (Skerra andPluckthun, Science 240:1038-1043 (1988)), beta-lactamase (Zemel-Dreasenand Zamir, Gene 27:315-322 (1984)), and those described in Abelson, J.and Simon, M. (eds), Methods in Enzymology, combinatorial chemistry,Vol. 267, San Diego: Academic Press (1996), and Kay, B. K., Winter, J.,McCafferty, J. (eds), Phage Display of peptides and Proteins, aLaboratory Manual, San Diego: Academic Press (1996), each incorporatedherein by reference.

[0329] Generally, the successful cloning strategy utilizing a phage coatprotein, such as pIII of filamentous phage fd, will provide: (1)expression of a protein chain (or a first polypeptide chain when thedesired protein is multichained, e.g., the V_(H) chain) fused to theN-terminus of a full sized (or nearly full sized) coat protein (e.g.,pIII) and transport to the inner membrane of the host where thehydrophobic domain in the C-terminal region of the coat protein anchorsthe fusion protein in the membrane, with the N-terminus containing thechain protruding into the periplasmic space and available forinteraction with a second or subsequent chain (e.g., V_(L) to form anFab fragment) which is thus attached to the coat protein; and (2)adequate expression of a second or subsequent polypeptide chain ifpresent (e.g., V_(L)) and transport of this chain to the solublecompartment of the periplasm.

[0330] In one embodiment for affinity enrichment of desired clones,about 10³ to 10⁴ library equivalents (a library equivalent is one ofeach recombinant—10⁴ equivalents of a library of 10⁹ members is10⁹×10⁴=10¹³ phage particles or phage) are incubated with target towhich the desired specific binding pair member (e.g., antibody) issought. The target is in one of several forms appropriate for affinityenrichment schemes. In one example the target is immobilized on asurface or particle, optionally anchored by a tether of enough length (3to 12 carbons, for example) to hold the target far enough away from thesurface to permit free interaction with the antibody combining site. Thelibrary of phage particle or phage bearing antibodies is then panned onthe immobilized target generally according to procedures well-known inthe art, for example, those described in Abelson, J. and Simon, M.(eds), Methods in Enzymology, combinatorial chemistry, Vol. 267, SanDiego: Academic Press (1996), Kay, B. K., Winter, J., McCafferty, J.(eds), Phage Display of peptides and Proteins, a Laboratory Manual, SanDiego: Academic Press (1996), each incorporated herein by reference.

[0331] A second example of target presentation is target attached to arecognizable ligand (again optionally with a tether of some length). Aspecific example of such a ligand is biotin. The target, so modified, isincubated with the library of phage particles or phage and bindingoccurs with both reactants in solution. The resulting complexes are thenbound to streptavidin (or avidin) through the biotin moiety. Thestreptavidin may be immobilized on a surface such as a plastic plate oron particles, in which case the complexes are physically retained; orthe streptavidin may be labelled, with a fluorophore, for example, totag the active phage/antibody for detection and/or isolation by sortingprocedures, e.g., on a fluorescence-activated cell sorter.

[0332] In one embodiment, the phage particles or phage bearingantibodies without the desired specificity are removed by various means,for example, by washing. The degree and stringency of washing requiredwill be determined for each specific binding pair member of interest. Acertain degree of control can be exerted over the bindingcharacteristics of the antibodies recovered by adjusting the conditionsof the binding incubation and the subsequent washing. The temperature,pH, ionic strength, divalent cations concentration, and the volume andduration of the washing will select for antibodies within particularranges of affinity for the hapten. Selection based on slow dissociationrate, which is usually predictive of high affinity, is the mostpractical route. This may be done either by continued incubation in thepresence of a saturating amount of free hapten, or by increasing thevolume, number, and length of the washes. In each case, the rebinding ofdissociated antibody-phage is prevented, and with increasing time,antibody-phage of higher and higher affinity are recovered.

[0333] Antibodies with certain catalytic activities may be enriched ingroups of antibodies with high affinity for reactants (substrates andintermediates) but low affinity for products. A double screen to enrichfor antibodies with these characteristics may be useful in findingantibodies to catalyze certain reactions. Further, catalytic antibodiescapable of certain cleavage reactions may also be selected. One categoryof such reactions is the cleavage of a specific end group from amolecule. For example, a catalytic antibody to cleave a specific aminoacid from an end of a peptide may be selected by immobilizing thepeptide and panning the antibody library under conditions expected topromote binding but not cleavage (e.g., low temperature, particularionic strength, pH, cation concentration, etc., depending on the natureof the end group and the cleavage reaction) and followed by a wash. Thisallows antibodies that recognize the end group to bind and becomeimmobilized, and from this group will come those capable of cleavage. Tofind those capable of cleavage, the conditions are shifted to thosefavorable for cleavage. This step will release those antibody-phagecapable of cleaving themselves free of the immobilized peptide.

[0334] An alternative way to accomplish this is to pan for antibodiesthat bind to the specific end group by attaching that end group to abond different from that to be cleaved (a non-peptide bond, forexample). By subsequent panning (of the positive phage from the firstscreen) on the end group attached via the proper bond under cleavageconditions, the non-binding fraction will be enriched for those with thedesired catalytic activity.

[0335] To elute the active antibody-phage particle or phage from theimmobilized target, after washing at the appropriate stringency, thebound (active) phage particle or phage can be recovered by eluting withpH shift. For example, pH2 or pH11 may be used, which is thenneutralized and the eluted phage are amplified by infecting ortransforming the host cells. The cells are then grown as tetracyclineresistant colonies. The colonies are scraped up and the extruded phageare purified by standard procedures as before. These phage are then usedin another round of affinity enrichment (panning), and this cycle isrepeated until the desired level of enrichment is reached or until thetarget phage are no longer enriched relative to the background phageparticles or phage. To isolate individual clones, phage particles orphage from the final round of panning and elution are infected intocells or their DNA is transformed into cells and grown on agar (usuallyL-agar) and antibiotics (usually tet) to form well separated individualcolonies, each of which is a clone carrying vectors with both V_(H) andV_(L) sequences. The single stranded DNA from phage particles or phageextruded from each colony may be isolated and DNA coding for the V_(H)and V_(L) fragments sequenced. The replicative form of the phage DNA(double stranded) may be isolated by standard means and the DNA in thecloning sites (V_(H) and V_(L) sequences) recloned into a vectordesigned for gene product expression in prokaryotes or eukaryotes toobtain larger amounts of the particular antibodies selected in thescreening process.

[0336] Phage identified as having an antibody recognized by the targetligand are propagated as appropriate for the particular phage vectorused. For fd-tet this is done in a liquid culture of rich medium(L-broth, for example) with antibiotic (Tet) selection. The phage areharvested and DNA prepared and sequenced by standard methods todetermine the DNA and amino acid sequence of the particular antibody.

[0337] The DNA may be recloned in a suitable eukaryotic or prokaryoticexpression vector and transfected into an appropriate host forproduction of large amounts of protein. Antibody is purified from ” theexpression system using standard procedures. The binding affinity of theantibody is confirmed by well known immunoassays with the target antigenor catalytic activity as described in Harlow and Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor, N.Y. (1988), Abelson, J. andSimon, M. (eds), Methods in Enzymology, combinatorial chemistry, Vol.267, San Diego: Academic Press (1996), Kay, B. K., Winter, J.,McCafferty, J. (eds), Phage Display of peptides and Proteins, aLaboratory Manual, San Diego: Academic Press (1996), each incorporatedherein by reference.

[0338] In another embodiment, phage particles or phage displaying thedesired specific binding pair member are affinity purified as follows:approximately 10³-10⁴ library equivalents of phage particles or phageare reacted overnight with 1 micropgram purified antibody at 4° C. Themixture is panned by a procedure as follows. A polystyrene petri plateis coated with 1 ml of streptavidin solution (1 mg/ml in 0.1M NaHCO₃, pH8.6, 0.02% NaN₃) and is incubated overnight at 40C. The following daythe streptavidin solution is removed. The plate is filled with 10 mlblocking solution (30 mg/ml BSA, 3 micrograms/ml streptavidin in 0.1MNaHCO₃₁ pH 9.2, 0.02% NaN₃) and incubated for 2 hours at roomtemperature. Two micrograms of biotinylated goat anti-mouse IgG (BRL)are added to the antibody-reacted library and incubated for 2 hours at40C. Immediately before panning, blocking solution is removed fromstreptavidin coated plate, and the plate is washed 3 times withTBS/0.05% Tween 20. The antibody-reacted library is then added to theplate and incubated for 30 minutes at room temperature. Streptavidincoated agarose beads (BRL) may also be used for this affinitypurification. The library solution is removed and the plate is washedten times with TBS/0.05% Tween 20 over a period of 60 minutes. Boundphage are removed by adding elution buffer (1 mg/ml BSA, 0.1N HCl, pHadjusted to 2.2 with glycine) to the petri plate and incubating for 10minutes to dissociate the immune complexes. The eluate is removed,neutralized with 2M Tris (pH unadjusted) and used to infect log phaseF′-containing bacterial cells. These cells are then plated on LB agarplates containing tetracycline (20 .mu.g/ml), and grown overnight at370C. Phage—particles or phage are isolated from these plates asdescribed and the affinity purification process was repeated for two tothree rounds. After the final round of purification, a portion of theeluate is used to infect cells and plated at low density on LBtetracycline plates. Individual colonies are transferred to culturetubes containing 2 ml LB tetracycline and grown to saturation. Phage orphagemid DNA is isolated using a method designed for the Beckman BiomekWorkstation (Mardis and Roe., Biotechniques, 7:840-850 (1989)) whichemploys 96-well microtiter plates. Single stranded DNA is sequenced bythe dideoxy method using Sequenase (U.S. Biochemicals) and anoligonucleotide sequencing primer (5′-′CGATCTAAAGTTTTGTCGTCT-3′) whichis complementary to the sequence located 40 nucleotides 3′ of the secondBstXI site in fdTetB1.

[0339] We considered a number of variables to address in theconstruction of a novel, very large antibody phage library: (i) theprimer design was optimised for amplification of variable gene pools tomaintain maximum diversity; (ii) a highly efficient two-step cloningmethod was developed to obtain a very large naive library; (iii) anantibody format and compatible cloning vector were chosen, which shouldpermit the rapid down-stream analysis of selected clones.

[0340] In order to achieve access to as many different human heavy andlight chain V-region gene segments as possible, a new set ofoligonucleotide primers was developed (Table I), the design of which wasbased on the most recent sequence information provided by the V-base.

[0341] The primers were designed to be the or several consensussequences which would have at least a 70% homology to the respective 5′or 3′ end based coding region in the human germ line gene segments ofthe specific V gene family they would have to amplify. The primers wouldamplify at least one V-gene segment using the PCR conditions describedbelow, and in one embodiment are appended with appropriate positionedrestriction sites for cloning into the vector for Fab expression.

[0342] The primers should allow efficient amplification of all commonlyused V-gene segments. Further, to obtain the large sized Fab librariesof the invention (over 10¹⁰ in diversity), we used a two-step cloningprocedure: heavy and light chain variable genes were first separatelycloned as digested PCR products, and were then combined by restrictionfragment cloning to form a large library of Fab fragments. This cloningprocedure should be a more efficient route for library construction thanthe relatively inefficient direct cloning of digested PCR-products,while avoiding the DNA instability often associated with in vivorecombination systems (Griffiths, et al., (1994) EMBO J. 13, 3245-3260).

[0343] A new phagemid vector, pCES1 (FIG. 1), was constructed, thatallows the stepwise cloning of antibody fragments in Fab format. In thisvector system, the variable heavy chain region genes are cloned asV_(H)-gene fragments; the vector supplies all Fab's with a human gamma-1C_(H1) domain. The V_(H)C_(H1) formed by insertion of the V_(H)-genefragments to the vector is fused (in the vector) to two tags forpurification and detection (a histidine tail for Immobilised MetalAffinity Chromatography (Hochuli, et al., (1988) BioTechnology 6,1321-1325) and a c-myc-derived tag (Munro, et al., H. R. (1986) Cell 46,291-300)), followed by an amber stop codon (Hoogenboom, et al., (1991)Nucleic Acids Res. 19, 4133-4137) and the minor coat protein III offilamentous phage f_(d). The antibody light chain is cloned as fullV_(L)C_(L) fragment, for directed secretion and assembly with theV_(H)C_(H1) on the phage particle.

[0344] In one embodiment, the vector comprises an expression cassettewith a bicistronic or double cistronic expression cassette to allowlinked (for the bicistronic) or independent (for the double cistronic)expression of the antibody light and heavy chain or their fusions, suchexpression cassette consisting of the following elements : (1) apromoter suited for non-inducible and inducible expression (e.g. lacZ);(2) a ribosome binding site and signal sequence preceeding the light andheavy chain cloning regions; (3) possible, but not necessarily, a regionfollowing the heavy or light chain cloning region that encodes a tagsequence such as a stretch of 5-6 hsitidines or a sequence recognised byan antibody and an amber codon; (4) a phage coat protein encoded as afusion to the 3′ end of either the heavy or light chain.

[0345] This new phage library will be a valuable source of antibodies toessentially any target. The antibodies may be used as research reagentsor as starting point for the development of therapeutic antibodies oragricultural products. As the list of sequenced genomes anddisease-related gene products is expanding rapidly, there will be agrowing need for an in vitro and eventually automated method forantibody isolation. As antibodies have been and will be ideal probes forinvestigating the nature, localization and purification of novel geneproducts, this library is envisaged to play an important role in targetvalidation and target discovery in the area of functional genomics.

[0346] Protein variants expressed on the surface of bacteriophage havebeen selected on the basis of their affinity for ligand (antigen) usingchromatography, panning or adsorption to cells. Elution from affinitymatrices has been achieved by specific elution using the ligand (antigenor a related compound) or non-specific elution using, for example, 100mM triethylamine. Washing procedures remove nonspecifically bound phage.The phage binds to and is eluted from the matrix according to theaffinity or the nature of the binding interaction. Specifically elutedphage are then used to infect male E. coli cells expressing the F pilus,allowing recovery of phage containing DNA encoding proteins with thedesired binding characteristics.

[0347] Selection can be made not only on the basis of specificity, butalso on the basis of affinity. Separation is readily attainable byaffinity chromatography between phage expressing an antibody with adissociation constant of 10⁻⁸M and one with a dissociation constant of10⁻⁵M. Clackson, T. et al. (1991). Nature 352: 624-628. The isolation ofthe latter antibody from an immune repertoire demonstrates thatantibodies with affinities characteristic of the primary immune responsecan be isolated using phage technology.

[0348] Antibodies directed against cell surface antigens can also beisolated by selective adsorption of phage on the surface of cells.Similarly, it may be possible to incorporate negative selection withcells to remove undesired cross-reactivities with cell surface markers.As these are rather difficult and as yet poorly understood methods,methods based on the selection on purified antigen should be usedwhenever possible.

[0349] Any selection for binders within a population will automaticallytend to select for high affinity variants at the expense of the lower,enriching the high affinity population. This has been used to goodeffect recently in the isolation of high affinity human antibodies froma naive repertoire. Marks, J. D. et al. (1991) J. Mol. Biol. 222,581-597. For optimal selection, the antigen concentration should be lessthan the affinity constant. This should be borne in mind when isolatingan antibody with pre-defined characteristics. Further details on variousselection methods is given in the reviews in this manual.

[0350] With such large panels of antibodies isolated, it is useful tohave methods available to readily determine the kinetic parameters ofeach individual antibody-antigen interaction. We have shown that it isfeasible to rapidly and accurately determine the off-rate ofnon-purified antibodies in periplasmic fractions prepared from smallscale cultures using surface plasmon resonance. Using this method, aseries of tetanus toxoid specific Fab's showed a monophasicdissociation, which is expected for a truly monomeric Fab-fragmentbinding to a low density antigen surface. Using this off-rate screeningassay, we determined the off-rates for the best tetanus toxoid and MUC1specific Fab's to be in the order of 10⁻² to 10⁻⁴ s⁻¹.

[0351] We tested the integrity of selected Fab's obtained fromperiplasmic fractions using western blots. When incubated innon-reducing sample buffer, two products were detected with the 9E10antibody, which recognises the myc-tag at the end of the CH1 domain, themajor product is the intact Fab-molecule, in which an intermoleculardisulfide bridge covalently links heavy and light chain fragments; thelow molecular product is most likely derived from non disulfide bridgelinked heavy chains. Analysis with anti-light chain sera reveals asimilar pattern and shows that the clones use a nearly equal percentageof kappa and lambda chains (found in six and seven clones respectivelyof a total of 13 tested). Upon reduction of purified, functionalantigen-binding Fabs, equal amounts of heavy and light chain are seen,while under non-reducing conditions, the main product is represented bythe disulphide linked Fab-molecule, with an equal amount of thenon-covalently linked V_(H)C_(H1) and V_(L)C_(L) products visible.Production yields of selected hormone specific Fab's varied between 160μg and 1.43 mg Fab per liter culture, which was in the same range as wasfound for the unselected Fabs.

[0352] A panel of 14 antigen-specific Fab's was fully sequenced (3anti-MUC1 antibodies; 11 anti-gonadotropin antibodies). The heavy chaingenes are derived from the four largest V_(H) families (V_(H1), V_(H3),V_(H4) and V_(H6)); the V_(L) genes belong to one of four V_(κ)-familiesor one of three V_(λ)-families. Chain promiscuity is seen for theα-chain specific clone SC#4G, the α/β-LH specific clones LH#2H andLH#3G, and β-FSH specific clone FS#8B, which all used a highlyhomologous V_(κ2) light chain gene segment combined with different heavychain fragments. The 3 anti-MUC1 antibodies use heavy and light chaingenes derived from 2 different VH and V6 families; clone MUC#9 uses a VHwith a cross-over of 2 segments.

[0353] The present invention is further illustrated in the followingexamples. Upon consideration of the present disclosure, one of skill inthe art will appreciate that many other embodiments and variations maybe made in the scope of the present invention. Accordingly, it isintended that the broader aspects of the present invention not belimited to the disclosure of the following examples.

[0354] As source of lymphoid tissues we used peripheral bloodlymphocytes from 4 healthy donors and part of a tumor-free spleenremoved from a patient with gastric carcinoma. B lymphocytes wereisolated from 2-L of blood on a Ficoll-Pacque gradient. For RNAisolation, the cell pellet was immediately dissolved in 50 ml 8 Mguanidinium thiocyanate/0.1 M 2-mercaptoethanol (Chirgwin, et al.,(1979) Biochemistry 18, 5294-5299). Chromosomal DNA was sheared tocompletion by passing through a narrow syringe (1.2/0.5 mm gauge), andinsoluble debris was removed by low speed centrifugation (15 min 2,934×g at room temperature). RNA was pelleted by centrifugation through aCsCl-block gradient (12 ml supernatant on a layer of 3.5 ml 5.7 MCsCl/0.1 M EDTA; in total 4 tubes) during 20 h at 125,000× g at 20° C.in a SW41-rotor (Beckman). The yield of total RNA was approx. 600 μg.RNA was stored at −20^(E)C in ethanol.

[0355] From the spleen, 2 g of tissue was used for homogenisation with apolytron in 20 ml 8 M guanidinium thiocyanate/0.1 M 2-mercaptoethanol.The total volume was increased to 80 ml with guanidinium thiocyanatebuffer, and after passage through a narrow syringe for shearing andremoval of debris, RNA was pelleted as described before, except for 15 hat 85,000× g at 20EC in a SW28.1 rotor (12 ml supernatant on 3.5 ml 5.7M CsCl/0.1 M EDTA in 5 SW28.1 tubes). From 2 g of tissue, 3 mg of totalRNA was extracted.

[0356] Random primed cDNA was prepared with 250 μg PBL RNA, while in aseparate reaction 300 μg spleen RNA was used as template. RNA was heatdenatured for 5 min at 65° C. in the presence of 20 μg random primer(Promega), subsequently buffer and DTT were added according to thesuppliers instructions (Gibco-BRL), as well as 250 μM DNTP (Pharmacia),800 U RNAsin (40 U/μl; Promega) and 2,000 U MMLV-RT (200 U/μl;Gibco-BRL) in a total volume of 500 μl. After 2 h at 42° C., theincubation was stopped by a phenol/chloroform extraction; cDNA wasprecipitated and dissolved in 85 μl water.

[0357] Oligonucleotides used for PCR amplification of human heavy andlight chain V-regions are described in FIG. 2. IgM-derived heavy chainvariable regions were obtained by a primary PCR with an IgM constantregion primer. All primary PCRs were carried out with separate BACKprimers and combined FOR primers, to maintain maximal diversity. ThePCR-products were reamplified with a combination of JHFOR-primers,annealing to the 3′ end of V_(H), and Sfi-tagged VHBACK-primers,annealing to the 5′ end, and subsequently cloned as V_(H)-fragments. Thelight chain V-genes of the kappa and lambda families were obtained byPCR with a set of CKFOR- or CλFOR-primer annealing to the 3′ end of theconstant domain and BACK-primers, priming at the 5′ end of theV-regions. The DNA-segments were reamplified with primers tagged withrestriction sites and cloned as V_(κ)C_(κ)- and V_(λ)C_(λ)-fragments.

[0358] PCR was performed in a volume of 50 μl using AmpliTaq polymerase(Cetus) and 500 μM of each primer for 28 cycles (1 min at 94^(E)C, 1 minat 55^(E)C and 2 min at 72^(E)C), 9 separate IgM derivedVH-amplifications were generated with 2 μl random primed cDNA(equivalent to 6 μg PBL RNA or to 7 μg spleen RNA) as template for eachreaction. For the light chain families, 6 different V_(κ)C_(κ)-productsand 11 V_(λ)C_(λ)-products (C_(λ3)- and C_(λ7)-primers combined in eachreaction) were obtained. All products were purified from agarose gelwith the QIAex-II extraction kit (Qiagen). As input for reamplificationto introduce restriction sites, 100-200 ng purified DNA-fragment wasused as template in a 100 μl reaction volume. The large amount of input,ensuring the maintenance of variability, was checked by analysis of 4 λlof the “unamplified” PCR-mixture on agarose gel.

[0359] For the construction of the primary heavy chain and the twoprimary light chain repertoires, the PCR-products, appended withrestriction sites, were gel purified prior to digestion and thedifferent V_(H)-, V_(κ)- and V_(λ)-families combined into three groups.The V_(κ)C_(κ)- and V_(λ)C_(λ)-fragments were digested with ApaLI andAscI, and cloned into the phagemid vector pCES1. The V_(H)-fragments,1.5 μg in total, were digested with SfiI and BstEII and ligated in a100-200 μl reaction mixture with 9 U T₄-DNA ligase at room temperatureto 4 μg, gel-purified vector pUC119-CES1 (similar to vector pCES1, butwith the pIII gene deleted). The desalted ligation mixture for light orheavy chain pools was used for electroporation of the E. coli strainTG1, to create the one-chain libraries.

[0360] The Fab library was obtained by cloning of V_(H) fragments,digested from plasmid-DNA prepared from the heavy chain repertoires,into the plasmid collection containing the light chain repertoires.Plasmid DNA isolated from at least 3×10⁹ bacteria of the V_(H) librarywas digested with SfiI and BstEII for cloning in the vector that alreadycontained λ and κ light chain libraries. To retain clones with internalBstEII site in the Vλ_(this site is relatively frequent in some λgermline V-segments (Persic, et al., (1997) Gene 187, 9-18), and also inthe constant domain of one of the λ families), the cloning ofV_(H)C_(H1) in the λ light chain repertoire containing vector was alsocarried out using SfiI and NotI cloning sites, to create a lessrestriction-biased V_(λ)_libary.

[0361] The rescue of phagemid particles with helper phage M13-KO7 wasperformed according to (Marks, et al., (1991) J. Mol. Biol. 222,581-597) on 10-L scale, using representative numbers of bacteria fromthe library for inoculation, to ensure the presence of at least 10bacteria from each clone in the start inoculum. For selections, 10¹³cfu's (colony forming units) were used with antigens immobilised inimmunotubes (Maxisorp tubes, Nunc) (Marks, et al., (1991) J. Mol. Biol.222, 581-597) or with soluble biotinylated antigens (Hawkins, et al.,(1992b) J. Mol. Biol. 226, 889-896). The amount of the immobilisedantigens tetanus toxoid and the hapten phenyl-oxazolone (conjugated toBSA in a ratio of 17 to 1) was reduced 10-fold during subsequentselection rounds, starting at 100 μg/ml at round 1. Capture withbiotinylated antigen in solution was used for a 100-mer peptide encodingfive copies of the tandem repeat of MUC1 (Henderikx, et al., (1998)Cancer Res. 58, 4324-4332), or with human Chorionic Gonadotropin (hCG),human Luteinizing Hormone (hLH), human Follicle Stimulating Hormone(hFSH) and its chimeric derivative (hFSH-CTP, containing the carboxyterminal peptide from the hCG β-subunit fused to the β-subunit of hFSH).Antigens were biotinylated at a ratio of ten to twenty moleculesNHS-Biotin (Pierce) per molecule antigen according to the suppliersrecommendations. Unless stated otherwise, the antigens were used forselection at concentrations of 100 nM, 30 nM and 10 nM during round 1, 2and 3 respectively. For hFSH-CTP 50, 15 and 10 nM was used respectively;for MUC1 peptide, 500, 100, 20 and 5 nM was used.

[0362] Soluble Fab was produced from individual clones as describedbefore (Marks, et al., (1991) J. Mol. Biol. 222, 581-597). Culturesupernatants were tested in ELISA with directly coated antigen orindirectly captured biotinylated antigen via immobilised biotinylatedBSA-streptavidin. Tetanus toxoid and phOx-BSA were coated at 10 μg/ml in0.1 M NaHCO₃ pH 9.6 for 16 h at 4° C. For coating of hCG and hFSH-CTP aconcentration of 4 μg/ml in 50 mM NaHCO₃ pH 9.6 was used. For capture ofbiotinylated antigens, biotinylated BSA was coated at 2 μg/ml in PBSduring 1 h at 37° C. After 3 washes with PBS-0.1% (v/v) Tween 20 (PBST),plates were incubated during 1 h with streptavidin (10 μg/ml in PBS/0.5%gelatin) (Henderikx, et al., (1998) Cancer Res. 58, 4324-4332).Following washing as above, biotinylated antigen was added for anovernight incubation at 4° C. at a concentration of 0.5 μg/ml for MUC-1peptide, 3 μg/ml for hLH, and 0.6 μg/ml for hFSH (binding to hCG wastested with directly coated antigen). The plates were blocked during 30min at room temperature with 2% (w/v) semi-skimmed milk powder (Marvel)in PBS. The culture supernatant was diluted 1 or 5-fold in 2% (w/v)Marvel/PBS and incubated 2 h; bound Fab was detected with anti-mycantibody 9E10 (5 μg/ml) recognising the myc-peptide tag at thecarboxyterminus of the heavy Fd chain, and rabbit anti-mouse-HRPconjugate (DAKO) (Marks, et al., (1991) J. Mol. Biol. 222, 581-597).Following the last incubation, staining was performed withtetramethylbenzidine (TMB) and H₂O₂ as substrate and stopped by addinghalf a volume of 2 N H₂SO₄; the optical density was measured at 450 nm.Clones giving a positive signal in ELISA (over 2× the background), wereanalysed by BstNI-fingerprinting of the PCR-products obtained byamplification with the oligonucleotide primers M13-reverse andgeneIII-forward (Marks, et al., (1991) J. Mol. Biol. 222, 581-597).

[0363] Large-scale induction of soluble Fab fragments from individualclones was performed on 50 ml scale in 2× TY containing 100 μg/mlampicillin and 2% glucose. After growth at 37° C. to an OD₆₀₀ of 0.9,the cells were pelleted (10 min at 2,934× g) and resuspended in 2× TYwith ampicillin and 1 mM IPTG. Bacteria were harvested after 3.5 hgrowing at 30° C. by centrifugation (as before); periplasmic fractionswere prepared by resuspending the cell pellet in 1 ml ice cold PBS.After 2 to 16 h rotating head-over-head at 4° C., the spheroplasts wereremoved by two centrifugation steps: after spinning during 10 min at3,400× g, the supernatant was clarified by an additional centrifugationstep during 10 min at 13,000× g in an eppendorf centrifuge. Theperiplasmic fraction obtained was directly used for determination offine specificities by surface plasmon resonance or for western blotstudies.

[0364] For sequencing, plasmid DNA was prepared from 50 ml culturesgrown at 30° C. in LB-medium, containing 100 μg/ml ampicillin and 2%glucose, using the QIAGEN midi-kit (Qiagen). Sequencing was performedwith the thermocycling kit (Amersham) with CY5-labeled primers CHLFOR(5′-GTC CTT GAC CAG GCA GCC CAG GGC-3′) and M13REV (5′-CAG GAA ACA GCTATG AC-3′); samples were run on the ALF-Express (Pharmacia). V-genesequences were aligned to V-base (Tomlinson et al., V-BASE, MRC Centrefor Protein Engineering, 1997,http://www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html) or the SangerCentre (Sanger Centre Germline Query, 1997, http//www.sanger.ac.uk/DataSearch/gq-search.html).

[0365] An hCG-preparation purified from urine and immuno-affinitypurified recombinant hLH, hFSH and hFSH-CTP produced in CHO-cells(Matzuk, et al., (1989) J. Cell. Biol. 109, 1429-1438; Muyan, et al.,(1996) Mol. Endocrinol. 10, 1678-1687) were used for western blotstudies as was described (Moyle, et al., (1990) J. Biol. Chem. 265,8511-8518). Between 0.5 and 1 μg of each hormone was loaded per lane;proteins were diluted in non-reducing sample buffer and boiled during 5min or directly applied on gel without heat-treatment; proteins weretransferred to blotting membrane by electrotransfer. Blots weresubsequently incubated for 16 h at room temperature with a 10-folddiluted periplasmic fraction in PBS/4% Marvel. Bound Fab was detectedwith anti-myc antibody 9E10 (5 μg/ml) and 4,000-fold diluted anti-mousealkaline phosphatase-conjugate (Promega), using the substrates5-bromo-1-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium(NBT) (Boehringer Mannheim) for visualisation.

[0366] The specificity of the Fab's was further characterised by surfaceplasmon resonance (BIAcore 2000, Biacore). Recombinant hLH, hFSH and theurinary hCG were immobilised on the flow-cells of a CM-chip using theNHS/EDC-kit (Pharmacia), yielding a surface of 1906 RU for hLH, 1529 RUfor hFSH-and 1375 RU for hCG. Periplasmic fractions were dilutedthree-fold in Hepes Buffered Saline (HBS; 10 mM Hepes, 3.4 nM EDTA, 150mM NaCl, 0.05% (v/v) surfactant P20, pH 7.4) and analysed using a flowrate of 10 μl/min.

[0367] Fab's were obtained by refolding of the total bacterial proteinsfrom a 50 ml culture (de Haard, et al., (1998) Protein Eng.,11:1267-1276). Briefly, the pelleted cells from a 50 ml inducedbacterial culture were resuspensed in 8 ml 8 M urea (in PBS). Aftersonication, the mixture was rotated head over head for 30 min andinsoluble material was removed by centrifugation for 30 min at 13,000×g. The supernatant was dialysed against PBS with four buffer changes.Insoluble proteins were removed by centrifugation and the flow throughfraction, obtained by filtration through a 0.2 μm membrane, wasimmediately loaded on an hCG column (bed volume 0.3 ml). The columnmaterial was prepared by coupling 8.4 mg protein to one gram Tresylsepharose according to the suppliers instructions (Pierce). The column(1 ml column material) was washed with 10 volumes 100 mM Tris, 500 mMNaCl pH 7.5, subsequently with 10 volumes 100 mM Tris/500 mM NaCl pH 9.5and with 2 volumes 0.9% NaCl, bound Fab was eluted with two volumes 0.1M TEA and immediately neutralised with 0.5 volume 1M Tris pH 7.5. TheFab fraction was dialysed against PBS using a Microcon 30 spin dialysisfilter (Amicon). Finally, a gel-filtration analysis was carried out on aSuperdex 75HR column (Pharmacia). The yield was determined by measuringthe optical density at 280 nm (using a molar extinction coefficient of13 for Fab's).

[0368] The kinetics of binding were analysed by surface plasmonresonance on three different hCG surfaces (303 RU, 615 RU and 767 RUimmobilised, with 4955 RU BSA on a separate flow cell as a negativecontrol). Fab present in crude periplasmic extracts was quantified on ahigh density surface of purified anti-human-Fab polyclonal antibody(Pierce) as described (Kazemier, et al., (1996) J. Immunol. Methods 194,201-209). Anti-hCG Fab's controls were purified by affinitychromatography on hCG columns as described above and used to calibratethe system.

[0369] The Fab library was constructed in two-steps. In the first step,variable region gene pools were amplified from approx. 4×10⁸ B-cellsfrom the PBLs of four healthy donors, and, as a source of possibly moreheavily mutated IgM antibodies, from a segment of a (tumor-free) spleenremoved from a patient with gastric carcinoma, containing approximately1.5×10⁸ B-cells (Roit, et al., (1985) Immunology, Gower MedicalPublishing, Ltd., London). Only IgM-derived V_(H) segments wereamplified by using an amplification with an oligonucleotide primerlocated in the first constant domain of this isotype. These productswere cloned into phagemid vector pCES1 for V_(L), and in pUC119-CES1 forV_(H) (cloning was more efficiently in the smaller sized vector, inwhich gene III was deleted). The PBL and spleen derived V_(H), V_(κ) andV_(λ)-libraries were cloned separately to maintain diversity, to yieldone-chain libraries in size typical for libraries made by cloning ofPCR-fragments (Marks, et al., (1991) J. Mol. Biol. 222, 581-597):1.75×10⁸ individual clones for the heavy chain, 9.4×10⁷ clones forV_(κ), and 5.2×10⁷ clones for V_(λ). In the second step, the heavy chainfragments were digested from plasmid DNA isolated from the primary V_(H)repertoire, and cloned into the vector containing the light chainrepertoires (again separately for PBL and spleen derived repertoire).The libraries were combined using this efficient cloning procedure, tocreate a naVve Fab repertoire with 3.7×10¹⁰ individual clones (4.3×10¹⁰recombinant clones, 86% of which have a full-length Fab insert), with70% of clones harbouring a kappa light chain, 30% a lambda chain. All of20 clones with full length Fab insert tested scored positive in dot-blotanalysis with the 9E10 antibody to indicating an expression level ofsoluble Fab of at least 0.2 mg/L.

[0370] We evaluated the library by selection with different antigens.First, the results from three model antigens, the protein tetanustoxoid, the hapten 2-phenyloxazol-5-one (phOx) (Griffiths, et al.,(1984) Nature 312, 271-275, and the peptide MUC1, are discussed. Threerounds of biopanning on tetanus toxoid yielded a diverse set of ELISApositive Fab's, in a series of 47 tetanus toxoid binding Fab's, at least21 were different with regard to BstNI-fingerprint. Similarly, anextensive panel of phOx-specific Fab's was retrieved after three roundsof panning: at least 24 different clones were identified in a series of50 ELISA positive clones. Solution capture with biotinylated MUC1peptide resulted in the selection of 14 different antibody fragments outof 37 ELISA-positive clones selected after 3 rounds.

[0371] As a more stringent test panel of antigens to assay theperformance of the library, we chose to derive antibodies to threestructurally related glycoproteins: human Chorionic Gonadotropin (hCG),human Luteinizing Hormone (hLH) and human Follicle Stimulating Hormone(hFSH) (reviewed in (Cole, (1997) Clin. Chem. 43, 2233-2243)). Thesehormones are heterodimers sharing an identical α-chain with 92 aminoacid residues, but have β-subunits of different composition and length.The β-chain of hCG contains 145 amino acid residues, and the one fromhLH only 121 residues, the latter showing 85% homology to β-hCG. Theβ-chain of hFSH is only 111 amino acids and shares 36% of the residueswith hCG. Antibodies that specifically detect hCG have been usedextensively in pregnancy tests (Cole, (1997) Clin. Chem. 43, 2233-2243)and for cancer diagnosis (Masure, et al., (1981) J. Clin. Endocrinol.Metab. 53, 1014-1020; Papapetrou, et al., (1980) Cancer 45, 2583-2592).A large set of antibodies to these targets would extend the limitednumber of hormone specific antibodies (especially against hLH), obtainedusing the hybridoma technology (Cole, (1997) Clin. Chem. 43, 2233-2243).The human origin of the antibodies might be beneficial when using thesefor imaging or therapy of testicular and bladder cancer (Masure, et al.,(1981) J. Clin.Endocrinol. Metab. 53, 1014-1020; Papapetrou, et al.,(1980) Cancer 45, 2583-2592).

[0372] Selections were thus performed on biotinylated urinary hCG,recombinant hLH, hFSH and hFSH-CTP (the latter is a chimeric moleculecontaining the carboxy terminal peptide of β-hCG fused to the β-chain ofFSH (Fares, et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89,4304-4308)). The highest degree of enrichment in respect to the increasein the number of eluted phage particles in round 3 versus round 1 wasfound for hCG (10,000-fold), followed by hFSH-CTP (1,000-fold), hFSH(300-fold) and hLH (150-fold). Polyclonal phage of selected populationswere tested for binding using sensorchips containing immobilisedhormones (Schier, et al., (1996) Hum. Antibodies Hybridomas 7, 97-105).Polyclonal phage selected with hCG showed binding after rounds two andthree of selection to all three proteins, i.e., hCG, hLH, and hFSH, withthe strongest signal visible for hCG. Similar analysis of the polyclonalphage populations selected for three rounds on hFSH showed a dominanceof hFSH-specific binding, while selections on hFSH-CTP yielded bindersto both hFSH and hCG. Selections on hLH yielded antibodies reactive withhFSH and hCG. Thus, this polyclonal phage screening provides a rapidtest to check the overall quality of the clones in the selectedrepertoire, and may also be used to guide the choice of the conditionsfor the next selection round (Schier, et al., (1996) Hum. AntibodiesHybridomas 7, 97-105).

[0373] ELISA of monoclonal phage antibodies revealed that three roundsof selection with hCG indeed resulted in the isolation of a highpercentage (74%) of clones positive for the gonadotropin. 27% of theseclones were hLH cross-reactive; none were reactive against streptavidin.BstNI-fingerprint analysis of the ELISA-positive clones revealed a highdegree of diversity (8 different patterns). From a representativehCG-specific (coded CG#4F) and hLH cross-reactive (CG#5C) clone, thespecificity was tested in BIAcore using unpurified soluble Fabfragments. Clone CG#4F gave a high response on hCG, with no visiblebinding to either hLH or hFSH-CTP. In contrast, clone CG#5C bound to hCGand hLH, but not to hFSH-CTP. Western blots, with the different hormonesin non-reduced form, showed the specific recognition of the β-subunit ofhCG by clone CG#4F, while the cross-reactive clone CG#5C reacted withthe β-subunit of both hCG and hLH.

[0374] Selection with the hormone hLH resulted in the isolation ofhLH-specific and hCG cross-reactive clones. Examination of individualclones from selection round three in ELISA revealed a large fraction ofhLH specific clones (69%), and a minor group of cross-reactive clones(16%); no streptavidin reactive clones were selected. Within the groupof specific clones, a large array of different species (>21) could bediscriminated with fingerprint analysis; however, all cross-reactivespecies had a single pattern. The unique hLH specificity was confirmedfor representative clones LH#2H and LH#3G, shown in surface plasmonresonance; and on western blot. LH#3G only recognises the intactα/β-heterodimer of hLH. Two representative clones of a pan-reactiveantibody in ELISA, coded LH#1C and LH#3F, reacted in BIAcore withhFSH-CTP, hCG and hLH, and in western blot analysis with the α-chainsfrom all three hormones.

[0375] When hFSH was used as antigen during selection, 6 differentantibodies were isolated from the library, with one type, represented byclone FS#8B, dominating the selected population. This Fab onlyrecognised hFSH in BIAcore, and, as western blot analysis demonstrated,in particular its β-unit. Further, the specificity of an α-chain bindingclone, SC#2B, was confirmed in BIAcore and western blot.

[0376] Upon selection with FSH-CTP 7 different α-chain specific Fab'swere identified by fingerprint analysis, from which the clones codedSC#2B, SC#2F, SC#2G and SC#4G were examined in more detail. Immunoblotanalysis with the recombinant Fab as detecting antibody confirmed thea-chain specificity.

[0377] The affinities and off-rates of affinity purified hCG reactiveFab's LH#1C, SC#2B, LH#3F and CG#5C were determined. The off-rates formost Fab's were in the order of 10⁻² and 10⁻³ s⁻¹. The off-rate valuesobtained using crude periplasmic fractions were in good agreement withthe values found for the purified Fab's, validating the utility of theoff-rate screen with unpurified Fab fragments. The affinities, 23 nM and38 nM for the α-subunit specific antibody LH#1C and the β-subunithCG/hLH-cross reactive antibody CG#5C respectively, are comparable tothe affinity of antibodies selected from a murine immune phage antibodylibrary (H.d.H., B. Kazemier, et al., unpublished); the top affinity,2.7 nM for the α-chain specific Fab SC#2B, approaches the values of thebest anti-hCG monoclonal antibodies (H.d.H., B. Kazemier, et al.,unpublished).

[0378] The aim of this procedure is to select and enrich forphage-antibodies to an antigen coated on the surface of immunotubes. Theantigen is coated to the immunotube (e.g., a Nunc-immunotube) andincubated with the phage library. Non-bound phage are washed away andthe binding phage are eluted, therefore the phage library becomesenriched for phage antibodies that specifically bind the antigen.

[0379] The aim of this procedure is to biotinylate proteins or peptides.At neutral pH or above, primary amine-groups react with NHS-SS-Biotin,and N-hydroxysulfosuccimide is released. The N-terminal free NH₂-groupsas well as lysines (K) of the protein react with NHS-S-S-Biotin, in thispH range.

[0380] NHS-SS-Biotin is a unique biotin analog with an extended spacerarm of approximately 24.3 Å in length, the spacer arm of NHS-LC-Biotinis 22.4 Å. These long chain analogs reduce steric hindrances associatedwith binding of biotinylated molecules to avidin or streptavidin.

[0381] The presence of the S-S linker in NHS-S-S-Biotin enablesdisruption of binding using reducing agents (DTT, DTE,B-mercaptoethanol). NHS-LC-Biotin is used when biotinylatedprotein/peptide is needed that is not sensitive to reducing agents.

[0382] The aim of this procedure is to select phage antibodies against abiotinylated antigen. The selection is done in solution, and can be usedto select phage antibodies against antigens that are prone todenaturation when coated onto solid surfaces.

[0383] First the biotinylated antigen is incubated with the phageantibody library. After addition of the Dynabeads (Dynal) coated withstreptavidin, the biotin of the antigen-antibody-complex will bind tothe streptavidin. This Dynabead-antigen-antibody-complex is pulled outwith a magnet (e.g., a Dynal magnet) and therefore should contain thespecific antibodies.

[0384] The aim of this procedure is to select for those antibodies outof a library that bind to antigens present in the cell membrane, usingadherent growing cells or cells in suspension. The method can be usedfor selection of antibodies against targets expressed on (tumor) celllines.

[0385] By incubating whole cells, organelles, or membrane fractions witha high variety phage antibody repertoire, such as the Fab Libraries ofthe invention (concentrated by PEG precipitation), only (orpreferentially) relevant antibodies, to one of the molecules exposed onthe surface of the cellular membrane(s), will be retained while notbinding phage antibodies are separated from the antibodies bound to thecells, organelles or membrane fractions (by methods well known in theart for separating cells, organelles or membrane fractions frommolecules in solution). The retained phage population is enriched forthose clones which are specific for cell related molecules. In principlethe following factors will positively influence the enrichment ofindividual clones: Affinity, antigen abundance, and low toxicity of theantibody construct to TG1 host.

[0386] The aim of this procedure is to prepare soluble antibodyfragments from the periplasm of E. coli. In the periplasm there is: lessprotease activity, less contaminating proteins than in the cytoplasm orsupernatants, and the antibody is more concentrated. Therefore,periplasmic preparations are more stable and more pure than culturesupernatants.

[0387] As a consequence of induction of phagemid containing bacterialcultures in low glucose medium with IPTG, soluble antibody fragments areproduced and directed to the periplasm where they are concentratedwithin 4 hours. Overnight culturing in these circumstances will make thebacterial membrane leaky and antibodies will be found in thesupernatant. For preparation of periplasmic fractions, the bacterialcell wall is first lysed by cold osmotic shock (icecold TES) and thenrapidly diluted in a chilled solution of low osmotic strength (TES/H₂O).The EDTA makes the outer membrane more permeable, and the cold inhibitsprotease activity. Subsequently, the bacterial cells are spun down andthe supernatant then contains the periplasmic proteins.

[0388] The antibodies in the periplasmic fraction can be used as a‘crude extract’ or the antibodies can be purified by conventional meanswell known in the art, for e.g., those recited in Section 5.6 and 5.7.

[0389] The aim of this procedure is to purify antibodies labeled with aHis6 tag from periplasmic fractions of Fabs made as described in Example6.18.

[0390] Immobilized metal affinity chromatography (IMAC) for thepurification of recombinant 6× His-tagged proteins under nativeconditions: Recombinant histidine tagged proteins are captured on achelated metal containing resin through coordination of free N-atoms ofthe histidines to the metal (mostly Ni²⁺ or Co 2+). After washing awaycontaminating proteins and other cell constituents, the his-taggedprotein is specifically eluted from the resin with imidazol whichcompetes for the binding of histidine-residues to the metal ion.

[0391] The present invention is not to be limited in scope by theexemplified embodiments which are intended as illustrations of singleaspects of the invention, and compositions and methods which arefunctionally equivalent are within the scope of the invention. Indeed,numerous modifications and variations in the practice of the inventionare expected to occur to those skilled in the art upon consideration ofthe present preferred embodiments. Consequently, the only limitationswhich should be placed upon the scope of the invention are those whichappear in the appended claims.

[0392] All references cited within the body of the instant specificationare hereby incorporated by reference in their entirety.

1. A plurality of polynucleotides encoding a Fab library comprising aplurality of vector wherein the vector comprises: a first and secondcloning region, wherein each cloning region comprises at least one, forthe vector unique, restriction enzyme cleavage site, each cloning regionbeing 5′ flanked by a ribosome binding site and a signal sequence, apolynucleotide encoding an anchor region, located 3′ of the secondcloning region, a first and a second plurality of variablepolynucleotides, each encoding a complete antibody variable region orpart of an antibody variable region, possibly followed by a completeantibody constant region or part of an antibody constant region, thefirst plurality of variable polynucleotides being cloned into the vectorat the restriction enzyme cleavage site(s) of the first cloning region,the second plurality of variable polynucleotides being cloned into thevector at the restriction enzyme cleavage site(s) of the second cloningregion.
 2. Polynucleotide according to claim 1, wherein the firstplurality of variable polynucleotides are V_(L) polynucleotides, and thesecond plurality of variable polynuclotides are V_(H) polynucleotides.3. Polynucleotides according to any of the preceding claims, wherein thepolynucleotides encode at least 10⁹ different Fabs, preferably at least10¹⁰ different Fabs, most preferably at least 3.7×10¹⁰ different Fabs.4. Fab library, comprising: a plurality of vectors, wherein the vectorcomprises: a first and a second cloning region, wherein each cloningregion comprises at least one, for the vector unique, restriction enzymecleavage site, each cloning region being 5′ flanked by a ribosomebinding site and a signal sequence, a polynucleotide encoding an anchorregion, located 3′ of the second cloning region, a first and a secondplurality of variable polynucleotides, each encoding a complete antibodyvariable region or part of an antibody variable region, possiblyfollowed by a complete antibody constant region or part of an antibodyconstant region, the first plurality of variable polynucleotides beingcloned into the vector at the restriction enzyme cleavage site(s) of thefirst cloning region, the second plurality of variable polynucleotidesbeing cloned into the vector at the restriction enzyme cleavage site(s)of the second cloning region to form a plurality of fusionpolynucleotides encoding a plurality of fusion proteins, a plurality ofcapsid particles, wherein the plurality of vector containing the firstand second pluralities of variable polynucleotides is packaged into thecapsid particles, wherein at least some of the capsid particles displaythe fusion protein encoded by the vector packaged into the capsid on thesurface of the capsid.
 5. Method of making a plurality ofpolynucleotides encoding a Fab library, comprising the steps of:amplifying a first plurality of variable polynucleotides with a firstset of primers, amplifying a second plurality of variablepolynucleotides with a second set of primers, wherein each set ofprimers comprises oligonucleotides designed to be homologous to the 5′and 3′ end of variable polynucleotides encoding antibody variableregions or parts thereof, such that they can be used to amplify variablepolynucleotide pools from natural or synthetic sources of genes whileretaining all or part of the antibody's antigen combining site; cloningthe first and second plurality of variable polynucleotides into aplurality of vectors, wherein the vector comprises: a first and a secondcloning region, wherein each cloning region comprises at least one, forthe vector unique, restriction enzyme cleavage site, each cloning regionbeing 5′ flanked by a ribosome binding site and a signal sequence, apolynucleotide encoding an anchor region, located 3′ of the secondcloning region, wherein the first plurality of variable polynucleotidesis cloned into the restriction enzyme cleavage site(s) of the firstcloning region of the vector and the second plurality of variablepolynucleotides into the restriction enzyme cleavage site(s) of thesecond cloning region of the vector.
 6. Method according to claim 5,further comprising the steps of: cloning the second plurality ofvariable polynucleotides into a plurality of another vector, andexcising the variable polynucleotides from the vector with a restrictionenzyme.
 7. Method of making a Fab library, wherein the plurality ofvector containing the first and second pluralities of variablepolynucleotides, obtained according to claim 4 or 5, are packaged into aplurality of capsid particles.
 8. Method for obtaining a Fab clone withspecificity to a target, comprising the steps of: obtaining a library ofclaim 4, and selecting an antigen-binding Fab using in vitro selectionon immobilised or labeled antigen.
 9. Monoclonal Fab or polyclonalcollection of Fab clones comprising one clone, respectively a pluralityof clones obtained from a library of claim 4 that specifically bind(s)to the human glycoprotein polymorphic epithelial Mucin-1 (MUC1). 10.Vector as defined in any of the claims 1-3.